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Antígeno B7-H1/metabolismo , Regulación Leucémica de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Genes myc , Linfoma Anaplásico de Células Grandes/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor de Transcripción STAT3/fisiología , Transcripción Genética/fisiología , Quinasa de Linfoma Anaplásico , Antígeno B7-H1/genética , HumanosRESUMEN
Macroautophagy is a physiological process that is implicated in various pathological conditions, including neurodegenerative diseases and cancer. The execution of canonical autophagy is regulated by a core signaling cascade and it involves two well-characterized, ubiquitin-like conjugation systems-the Atg5/Atg12/Atg16L1 and the Atg8-phosphatidyl ethanolamine (PE), which are both catalyzed by Atg7. The conjugation of Atg5-Atg12 and the subsequent interaction with the positive regulator Atg16L1 are essential for the conjugation of Atg8 to PE and the subsequent formation of autophagosomes. The interaction between Atg5-Atg12 complex and Atg16L1 is highly dynamic, induced upon activation of the autophagic process, and required for the recruitment of the At5-Atg12 complex to sites of autophagosome formation. Monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation may be used not only as means to study the dynamics of autophagy, but in another important point, it may provide important insights on the basic molecular mechanisms of autophagy in physiological and pathological settings. In this chapter, we describe methods of monitoring the Atg5-Atg12-Atg16L1 aggregation and deaggregation, with emphasis on prostate cancer.
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Proteína 12 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Inmunoprecipitación/métodos , Autofagia , Línea Celular , Línea Celular Tumoral , Humanos , Masculino , Biología Molecular/métodos , Neoplasias de la Próstata/metabolismo , Agregación Patológica de Proteínas/metabolismoRESUMEN
Extracellular vesicles have gained tremendous attention in the recent years as a novel mechanism of cell to cell communication. There are several types of extracellular vesicles, including exosomes, microvesicles, exosome, like vesicles, apoptotic bodies that differ mainly in the mechanism of biogenesis and secretion. The most well studied type of extracellular vesicles are the exosomes which are endosome-derived vesicles with a diameter of 50-150nm and enriched in ESCRT proteins including Alix, TSG101, Hsp70, and tetraspanins. It is now well established that exosomes promote tumor growth, alter the tumor microenvironment, facilitate the dissemination of cancer cells in an organotropic manner, modulate immune responses, and mediate resistance to therapy. Exosomes have also been recently implicated in an emerging hallmark of cancer, the cancer cell metabolism. The metabolic state of the cell defines, to a certain extent, both the rate of secretion and the molecular content of tumor-derived exosomes. Furthermore, exosomes have been shown to possess intrinsic metabolic activity since they can synthesize ATP by glycolysis. It follows that exosomes carry a number of metabolic enzymes and metabolites, including lactate, PGE, LDH isoforms, pyruvate, and monocarboxylate transporters. Last but not the least, exosomes are implicated in fatty acid synthesis and cholesterol metabolism and are thought to be crucial for the transcellular metabolism procedure. Uptake of exosomes is thought to alter the intracellular metabolic state of the cell. In summary, we describe the state of the art on the role of metabolism in the secretion, uptake, and the biological effects of exosomes in the metabolism of recipient cells.
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Exosomas/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Animales , Exosomas/fisiología , Femenino , Humanos , Masculino , Neoplasias/fisiopatologíaRESUMEN
Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.
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Bencenosulfonatos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carcinoma , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Resistencia a Antineoplásicos/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Masculino , Terapia Molecular Dirigida , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Próstata , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal/genética , SorafenibRESUMEN
Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1-12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.
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Antraciclinas/uso terapéutico , Antineoplásicos/uso terapéutico , Calreticulina/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Calreticulina/efectos de los fármacos , Estudios de Cohortes , Células Dendríticas/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Inmunidad Celular/efectos de los fármacos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Fagocitosis/efectos de los fármacos , Fosforilación , Análisis de Supervivencia , Linfocitos T/inmunologíaRESUMEN
Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.
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Antineoplásicos Hormonales/farmacología , Apoptosis , Autofagia , Dexametasona/farmacología , Leucemia Linfoide/metabolismo , Adolescente , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/agonistas , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Morfolinas/farmacología , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína de la Leucemia Promielocítica , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/agonistas , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/agonistas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tumor endothelial cells have long been regarded as genomically stable and therefore less likely to develop resistance to antiangiogenic therapies. However, recent findings have challenged this notion. We have shown that DNA can be transferred between cells through phagocytosis of apoptotic bodies by adjacent viable cells. Propagation of the ingested DNA is prevented by the activation of the p53-p21 pathway. In this study, we examined whether concomitant transfer of tumor DNA with genes that inactivate the p53 pathway could overcome the barrier to tumor DNA propagation. Our results demonstrate that fibroblasts and endothelial cells are capable of acquiring and replicating tumor DNA when the apoptotic tumor cells contain the SV40 large T antigen. Analysis of the tumor stroma of xenotransplanted tumors in severe combined immunodeficient mice revealed that a sub-population of the endothelial cells contained tumor DNA. These cells maintained the ability to form functional vessels in an in vivo assay and concurrently express tumor-encoded and endothelial-specific genes.
Asunto(s)
ADN de Neoplasias/metabolismo , Transferencia de Gen Horizontal , Animales , Antígenos Transformadores de Poliomavirus/genética , ADN de Neoplasias/genética , Células Endoteliales/metabolismo , Humanos , Ratones , Fagocitosis , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The exposure of calreticulin (CRT) on the plasma membrane can precede anthracycline-induced apoptosis and is required for cell death to be perceived as immunogenic. Mass spectroscopy, immunofluorescence and immunoprecipitation experiments revealed that CRT co-translocates to the surface with another endoplasmic reticulum-sessile protein, the disulfide isomerase ERp57. The knockout and knockdown of CRT or ERp57 inhibited the anthracycline-induced translocation of ERp57 or CRT, respectively. CRT point mutants that fail to interact with ERp57 were unable to restore ERp57 translocation upon transfection into crt(-/-) cells, underscoring that a direct interaction between CRT and ERp57 is strictly required for their co-translocation to the surface. ERp57(low) tumor cells generated by retroviral introduction of an ERp57-specific shRNA exhibited a normal apoptotic response to anthracyclines in vitro, yet were resistant to anthracycline treatment in vivo. Moreover, ERp57(low) cancer cells (which failed to expose CRT) treated with anthracyclines were unable to elicit an anti-tumor response in conditions in which control cells were highly immunogenic. The failure of ERp57(low) cells to elicit immune responses and to respond to chemotherapy could be overcome by exogenous supply of recombinant CRT protein. These results indicate that tumors that possess an intrinsic defect in the CRT-translocating machinery become resistant to anthracycline chemotherapy due to their incapacity to elicit an anti-cancer immune response.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Calreticulina/metabolismo , Mitoxantrona/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/uso terapéutico , Calreticulina/genética , Calreticulina/fisiología , Línea Celular Tumoral , Membrana Celular/enzimología , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Mitoxantrona/uso terapéutico , Datos de Secuencia Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/fisiología , Transporte de ProteínasRESUMEN
Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activation), elements of the endoplasmic reticulum stress response (such as eukaryotic initiation factor 2alpha phosphorylation), as well as elements of the apoptotic response (such as caspase activation). Depending on the signal-transduction pathway, tumor cells responding to chemotherapy or radiotherapy can express 'danger' and 'eat me' signals on the cell surface (such as NKG2D ligands, heat-shock proteins and calreticulin) or can secrete/release immunostimulatory factors (such as cytokines and high-mobility group box 1) to stimulate innate immune effectors. Likewise, the precise sequence of such events influences the 'decision' of the immune system to mount a cognate response or not. We therefore anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.
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Apoptosis/inmunología , Muerte Celular/inmunología , Neoplasias/inmunología , Neoplasias/fisiopatología , Animales , Autofagia , Calreticulina/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Proteína HMGB1/inmunología , Proteína HMGB1/metabolismo , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Humanos , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Células Asesinas Naturales/inmunología , Necrosis , Neoplasias/patología , Fagocitosis , Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.
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Antraciclinas/farmacología , Calcio/metabolismo , Calreticulina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Apoptosis , Brefeldino A/farmacología , Línea Celular Tumoral , Células HeLa , Homeostasis , Humanos , Inhibidores de la Síntesis de la Proteína/farmacologíaAsunto(s)
Apoptosis/inmunología , Apoptosis/efectos de la radiación , Calreticulina/metabolismo , Rayos gamma/uso terapéutico , Terapia Ultravioleta/métodos , Animales , Calreticulina/genética , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Neoplasias del Colon/radioterapia , Fibrosarcoma/inmunología , Fibrosarcoma/patología , Fibrosarcoma/radioterapia , Inmunogenética , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying H(+)- and Na(+)-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5-10.0. In cells grown at pH 4.5, inorganic phosphate (P(i)) was accumulated by two kinetically discrete H(+)/P(i)-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P(i) concentrations. The high-affinity system, subjected to regulation by both extracellular P(i) availability and intracellular polyphosphate stores, is mobilized during P(i)-starvation. In cells grown at pH 9.5-10, P(i) uptake is mediated by several kinetically discrete Na(+)-dependent systems that are specifically activated by Na(+) ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P(i) concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P(i)-starvation and appear to be controlled by extracellular P(i). They represent the first examples of high-capacity, Na(+)-driven P(i) transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H(+)- and Na(+)-coupled P(i) transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H(+)-coupled P(i) transport systems are predominant. The contribution of the Na(+)/P(i) cotransport systems to the total cellular P(i) uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher.
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Simportadores de Protón-Fosfato/metabolismo , Simportadores/metabolismo , Ácidos , Álcalis , División Celular , Medios de Cultivo , Metabolismo Energético , Concentración de Iones de Hidrógeno , Fosfatos/metabolismo , Fosfatos/farmacología , Proteínas Cotransportadoras de Sodio-Fosfato , Yarrowia/efectos de los fármacos , Yarrowia/crecimiento & desarrollo , Yarrowia/metabolismoRESUMEN
We have previously shown that one of the most potent rodent hepatocarcinogens, perfluorooctanoic acid (PFOA), induces apoptosis in human HepG2 cells in a dose- and time-dependent manner. In this study we have investigated the involvement of reactive oxygen species (ROS), mitochondria, and caspase-9 in PFOA-induced apoptosis. Treatment with 200 and 400 microM PFOA was found to cause a dramatic increase in the cellular content of superoxide anions and hydrogen peroxide after 3 h. Measurement of the mitochondrial transmembrane potential (Delta Psi(m)) after PFOA treatment showed a dissipation of Delta Psi(m) at 3 h. Caspase-9 activation was seen at 5 h after treatment with 200 microM PFOA. In order to evaluate the importance of these events in PFOA-induced apoptosis, cells were cotreated with PFOA and N-acetylcysteine (NAC), a precursor of glutathione, or Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT pore). NAC reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced ROS. In addition, CsA also reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced opening of the MPT pore. In summary, we have delineated a ROS and mitochondria-mediated pathway for induction of apoptosis by PFOA.
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Apoptosis/efectos de los fármacos , Caprilatos/farmacología , Carcinoma Hepatocelular/patología , Fluorocarburos/farmacología , Neoplasias Hepáticas/patología , Mitocondrias Hepáticas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Carcinógenos/farmacología , Caspasa 9 , Caspasas/metabolismo , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/metabolismo , Cinética , Potenciales de la Membrana , Superóxidos/metabolismo , Células Tumorales CultivadasRESUMEN
The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50-150 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 microM PFOA, prior to nuclear fragmentation.