RESUMEN

Cytokines play an important role in the immune system, and abnormalities in their production have been found in many human diseases. Interleukin-21 (IL-21), a type I cytokine produced by activated T cells, has diverse effects on the immune system, but its ability to induce production of other cytokines is not well delineated. Furthermore, the signaling pathway underlying its action is poorly understood. Here, we have evaluated IL-21-induced cytokine production in human monocytes and U937 leukemia cells. We found that IL-21 induces upregulation of a variety of cytokines from multiple cytokine families. We also found that IL-21 triggers rapid activation of ERK1/2. Neutralizing antibody to the IL-21R prevented both IL-21-induced cytokine production and IL-21-induced activation of ERK1/2. Inhibition of ERK1/2 activity by the ERK-selective inhibitor U0126 reverses the ability of IL-21 to upregulate cytokine production, suggesting that IL-21-induced cytokine production is dependent on ERK1/2 activation.

Asunto(s)

Citocinas/biosíntesis , Interleucinas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Butadienos/farmacología , Activación Enzimática , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Monocitos/metabolismo , Nitrilos/farmacología , Transducción de Señal , Células U937 , Regulación hacia Arriba

RESUMEN

PURPOSE: To evaluate the novel taxane analogs, BMS-184476 and BMS-188797, as potential radiosensitizers in vitro and in vivo. METHODS AND MATERIALS: Human H460 lung cancer cells were incubated with either paclitaxel or a taxane analog and irradiated at various times. Surviving fractions were then determined using a clonogenic assay. Three different schedules were used: (A) 1-h drug incubation with radiation at t = 8 h, (B) 1-h drug incubation with radiation at t = 24 h, (C) 24-h drug incubation with radiation immediately after. Cell cycle redistribution by taxanes alone was measured with propidium iodide and flow cytometry. Percent apoptosis was also measured using 7-aminoactinomycin D (7-AAD) staining with flow cytometry. For in vivo studies, H460 cell xenografts were used in nude mice. Tumors were grown s.c. on the flank and then treated with BMS-184476 (10 mg/kg i.p. injection, Days 0, 2, and 4) and/or radiation (2 Gy/day, Days 0-4). Tumor growth delay was then measured for each treatment group. RESULTS: The mean in vitro radiation dose enhancement ratios of BMS-184476, BMS-188797, and paclitaxel were 1.76, 1.49, and 1.31 for Schedules A, B, and C, respectively. Isobologram analysis showed that BMS-184476 was synergistic with radiation using Schedule A. Treatment with taxanes caused an increase in the percentage of G2/M cells at the time of irradiation. The mean fold increases in the %G2/M above control values for all three drugs were 5.6, 2.5, and 1.7 for Schedules A, B, and C, respectively. The combined effects of taxanes plus radiation on the induction of apoptosis were additive for all three drugs. In vivo studies showed that BMS-184476 can enhance the effects of fractionated radiotherapy, with an average enhancement factor of 1.66 obtained from three independent experiments. CONCLUSIONS: These results demonstrated that the novel taxane analogs, BMS-184476 and BMS-188797, can enhance the effects of radiation in human lung cancer cells both in vitro and in vivo. These data also support the hypothesis that a G2/M block is involved in the radiosensitization caused by the taxanes.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Paclitaxel/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Taxoides , Apoptosis , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Humanos , Interfase/efectos de los fármacos , Interfase/efectos de la radiación , Paclitaxel/análogos & derivados , Células Tumorales Cultivadas

RESUMEN

Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mitógenos/farmacología , Mutación , Animales , Células CHO , Dominio Catalítico/genética , Cricetinae , Dimerización , Proteína Adaptadora GRB2 , Transferasas Intramoleculares/metabolismo , Mitosis , Fosforilación , Transporte de Proteínas , Proteínas/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Rubidio/metabolismo , Transducción de Señal , Activación Transcripcional

RESUMEN

Deficiency of folate or vitamin B(12) (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts from p53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Asunto(s)

Anemia Megaloblástica/genética , Apoptosis , Ciclo Celular/fisiología , Replicación del ADN , Eritroblastos/fisiología , Genes p53 , Anemia Megaloblástica/patología , Anemia Megaloblástica/fisiopatología , Animales , Células Cultivadas , Reparación del ADN , Eritroblastos/patología , Ácido Fólico/metabolismo , Cinética , Ratones , Ratones Endogámicos , Ratones Noqueados , Fase S , Vitamina B 12/metabolismo

RESUMEN

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

Asunto(s)

Proteínas de Ciclo Celular , Receptores ErbB/antagonistas & inhibidores , Fase G1/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/biosíntesis , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Supresoras de Tumor , Animales , Ciclina D1/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Quinazolinas/farmacología , Proteína de Retinoblastoma/metabolismo , Tirfostinos/farmacología , Regulación hacia Arriba

RESUMEN

Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.

Asunto(s)

Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Eritroblastos/patología , Eritroblastos/fisiología , Eritropoyetina/farmacología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Animales , Línea Celular , Supervivencia Celular/fisiología , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

We have examined the effect of neutralizing TGF-beta antibodies on cisplatin-mediated cytotoxicity against MDA-231 human breast tumor cell spheroids. These tridimensional in vitro systems have been shown to recapitulate the drug sensitivity pattern of tumor cells in vivo. MDA-231 tumor cell spheroids exhibit higher protein levels of the cyclin-dependent kinase (Cdk) inhibitors p21 and p27 and >10-fold lower Cdk2 activity compared to adherent cell monolayers, as well as pRb hypophosphorylation, a predominant G1 population, and a cisplatin 1-h IC50 of approximately 100 microM. Treatment of MDA-231 cells in monolayer with cisplatin for 1 h, subsequently grown as spheroids, increased steady-state TGF-beta1 mRNA levels, secretion of active TGF-beta, cellular Cdk2 activity, pRb phosphorylation, and p21 protein levels, while downregulating p27. Accumulation of cells in G2M and progression into S were noted 48 h after treatment with 100 microM cisplatin. We tested whether drug-induced upregulation of TGF-beta1 and p21, perhaps by preventing cell cycle progression, were protective mechanisms against drug-mediated toxicity by using neutralizing anti-TGF-beta antibodies. Anti-TGF-beta antibodies diminished the induction of p21, enhanced the activation of Cdk2, and facilitated progression into S and G2M following cisplatin treatment. This resulted in a >twofold enhancement of drug-induced DNA fragmentation and a shift in the cisplatin 1-h IC50 from 100 to <10 microM. These data suggest that tumor cell TGF-beta1 may protect from DNA damage and that postchemotherapy administration of TGF-beta inhibitors may facilitate progression beyond G1/S, potentially increasing the efficacy of cytotoxic chemotherapy.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Ciclo Celular/efectos de los fármacos , Cisplatino/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Proteínas Supresoras de Tumor , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Agregación Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Cisplatino/uso terapéutico , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina G/farmacología , Concentración 50 Inhibidora , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales Cultivadas

RESUMEN

Dopamine (DA) and related catechols may contribute to selective degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease. To investigate whether DA induces apoptosis of dopaminergic neurons, we characterized the effects of various concentrations of exogenous DA on a substantia nigra/neuroblastoma hybrid cell line (MES 23.5 or MES). The hybrid MES cells were maintained in the presence of 50 microM glutamate in logarithmic growth on poly-D-lysine-precoated T-75 flasks and plated either onto petri dishes with glass coverslips for morphological studies or onto 6-well plates for quantification of apoptosis by flow cytometry. The results showed that DA exposure (0.5-20 microM) induced time- and dose-dependent apoptotic cell death of MES cells. To further analyze the mechanism responsible for DA-mediated apoptosis, we repeated the experiments at 20 microM DA in the presence or absence of 40 microM nomifensine, a DA re-uptake inhibitor, and 25 microM 2-amino-5-phosphonopentanoic acid (AP5), an N-methyl-D-aspartate (NMDA) receptor antagonist. The data indicate that both compounds significantly prevented DA-induced apoptosis of MES cells and that combination of AP5 and nomifensine provided greater protection against DA toxicity than AP5 alone. These results suggest for the first time that DA-induced apoptosis in dopaminergic neurons is partially attributable to increased vulnerability of these cells to non-toxic levels of excitatory amino acids, i.e., secondary excitotoxicity.

Asunto(s)

Apoptosis , Dopamina/farmacología , Neuronas/citología , Neuronas/fisiología , Neurotoxinas/farmacología , Sustancia Negra/citología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Células Híbridas , Cinética , Neuroblastoma , Nomifensina/farmacología , Polilisina , Células Tumorales Cultivadas

RESUMEN

The incidence of DNA mutation and subsequent risk of transformation in different cell types may depend on cell type-specific variation in position and duration of cell cycle arrest after exposure to DNA-damaging agents. To determine whether cell type-specific checkpoints occur, normal human epidermal keratinocytes (HKs) and human dermal fibroblasts (HFs), isolated from the same tissue, were exposed to genotoxic agents. Following exposure, cell cycle arrest profiles, cell proliferation rates, and select protein levels and activities were analyzed and found to be cell type dependent. After exposure to either gamma-radiation or Adriamycin, HFs arrested primarily in G1, whereas HKs arrested predominantly in G2. The attenuated G1 arrest in the HKs correlated with less p53 protein accumulation, as compared to that observed in G1-arrested HFs. Although gamma-irradiated HFs were unable to reenter the cell cycle, HKs began proliferating 72 h posttreatment. Consistent with the cell cycle profiles observed, cyclin-dependent kinase activities were inhibited for a longer duration in HFs as compared to HKs after gamma-irradiation. The results indicate that cell cycle checkpoint response to genotoxic insult may vary according to cell type within any given tissue. The attenuated G1 arrest observed in HKs may be an important factor in the transforming events leading to skin neoplasia.

Asunto(s)

Quinasas CDC2-CDC28 , Ciclo Celular/fisiología , Fibroblastos/citología , Queratinocitos/citología , Ciclo Celular/efectos de la radiación , Transformación Celular Neoplásica , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Fase G1/efectos de los fármacos , Fase G1/efectos de la radiación , Fase G2/efectos de los fármacos , Rayos gamma , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , Mutágenos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Interferon gamma (IFNgamma) inhibits the growth and differentiation of highly purified human erythroid colony-forming cells (ECFCs) and induces erythroblast apoptosis. These effects are dose- and time-dependent. Because the cell surface receptor known as Fas (APO-1; CD95) triggers programmed cell death after activation by its ligand and because incubation of human ECFCs with IFNgamma produces apoptosis, we have investigated the expression and function of Fas and Fas ligand (FasL) in highly purified human ECFCs before and after incubation with IFNgamma in vitro. Only a small percentage of normal human ECFCs express Fas and this is present at a low level as detected by Northern blotting for the Fas mRNA and flow cytometric analysis of Fas protein using a specific mouse monoclonal antibody. The addition of IFNgamma markedly increased the percentage of cells expressing Fas on the surface of the ECFCs as well as the intensity of Fas expression. Fas mRNA was increased by 6 hours, whereas Fas antigen on the cell surface increased by 24 hours, with a plateau at 72 hours. This increase correlated with the inhibitory effect of IFNgamma on ECFC proliferation. CH-11 anti-Fas antibody, which mimics the action of the natural FasL, greatly enhanced IFNgamma-mediated suppression of cell growth and production of apoptosis, indicating that Fas is functional. Expression of FasL was also demonstrated in normal ECFCs by reverse transcriptase-polymerase chain reaction and flow cytometric analysis with specific monoclonal antibody. FasL was constitutively expressed among erythroid progenitors as they matured from day 5 to day 8 and IFNgamma treatment did not change this expression. Apoptosis induced by IFNgamma was greatly reduced by the NOK-2 antihuman FasL antibody and an engineered soluble FasL receptor, Fas-Fc, suggesting that Fas-FasL interactions among the ECFCs produce the erythroid inhibitory effects and apoptosis initiated by IFNgamma.

Asunto(s)

Apoptosis , Eritrocitos/patología , Eritropoyesis , Interferón gamma/farmacología , Glicoproteínas de Membrana/fisiología , Receptor fas/fisiología , Animales , Apoptosis/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Proteína Ligando Fas , Humanos , Ratones , Proteínas Recombinantes

RESUMEN

Interferon gamma (IFNgamma) has been shown to inhibit proliferation and differentiation of erythroid progenitor cells and to produce apoptosis of erythroid cells, whereas stem cell factor (SCF), erythropoietin (EP), and insulin-like growth factor-I (IGF-I) have distinct roles in enhancing erythroid cell production and preventing apoptosis. The mechanism by which IFNgamma exerts an inhibitory effect on the positive roles of these growth factors is unknown. Although some inhibitory cytokines including IFNgamma have been shown to downregulate growth factor receptors, the effect of IFNgamma on SCF, EP, and IGF-I receptors of human erythroid progenitor cells has not been defined. We obtained highly purified day-5 or day-6 erythroid colony-forming cells (ECFCs) from human blood in sufficient quantity and purity for radiolabeled cytokine binding studies and analysis of mRNA. When day-5 ECFCs were incubated with increasing concentrations of recombinant human (rh) IFNgamma for 24 hours at 37 degrees C, specific binding of 125I-rhSCF to SCF receptors was significantly decreased by 25% to 40% in a dose-dependent fashion, with the maximum effect at 2,500 to 5,000 U/mL of IFNgamma. The decrease was apparent by 12 hours of incubation and was only slightly lower by 24 hours. The numbers of SCF and EP receptors, but not of IGF-I receptors, per ECFC, calculated by Scatchard analysis, were significantly decreased by 30% and 23% to 25%, respectively, after incubation with 2,500 U/mL rhIFNgamma for 24 hours at 37 degrees C, whereas the binding affinities were not affected. This decrease in SCF receptors was confirmed by flow cytometry using an anti-c-kit mouse monoclonal antibody. Northern blot analysis showed that the mRNAs for the SCF and EP receptors, but not for the IGF-I receptors, were decreased by 50% to 60% after 3 hours of incubation at 37 degrees C with 2,500 U/mL of rhIFNgamma. This persisted for 24 hours without alteration of the stability of the SCF and EP receptor mRNAs. These observations suggest that one means by which IFNgamma inhibits erythroid cell proliferation and differentiation and produces apoptosis may be through the reduction of the number of target receptors for SCF and EP and that this occurs through transcriptional inhibition of the corresponding mRNAs.

Asunto(s)

Células Precursoras Eritroides/metabolismo , Eritropoyetina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interferón gamma/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores de Eritropoyetina/metabolismo , Factor de Células Madre/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , Proteínas Recombinantes

RESUMEN

BACKGROUND: Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R. PURPOSE: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. METHODS: MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R content was assessed by 125I-EGF binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ. Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA. RESULTS: Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells. By use of a colony-forming assay, the 1-hour IC50 (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin. Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones. CONCLUSIONS: These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/fisiopatología , Cisplatino/farmacología , Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , ARN sin Sentido , Transducción de Señal , Animales , Aductos de ADN , ADN Nucleotidilexotransferasa , ADN Complementario , ADN de Neoplasias/efectos de los fármacos , Receptores ErbB/genética , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante Heterólogo , Células Tumorales Cultivadas

RESUMEN

The targeted disruption of the low density lipoprotein (LDL) receptor gene in mice results in accumulation of plasma LDL cholesterol and in predisposition to diet-induced aortic atherosclerosis. Although the liver is the central organ for receptor mediated clearance of LDL, the in vivo role of other organs and tissues in LDL catabolism has not been directly studied. Since bone marrow-derived cells such as blood leukocytes and tissue macrophages express LDL receptors and contribute a large mass to the body, we designed bone marrow transplantation (BMT) experiments to reconstitute LDL receptor null mice [LDL-R(-/-)] with marrow obtained from LDL-R wild-type mice [LDL-R(+/+)] and evaluate the effects on parameters of plasma lipid metabolism. Although reconstitution of the transplanted mice with donor bone marrow cells was complete, no differences in plasma lipid levels and lipoprotein distribution were found between groups, irrespective of the diet used, and turnover studies using 125I-labeled LDL showed that LDL receptor expression by leukocytes and macrophages does not significantly contribute to plasma LDL clearance. The complementary experiment of transplanting LDL-R(-/-) marrow into C57BL/6 recipients [LDL-R(-/-)-->LDL(+/+)], performed to evaluate the role of leukocyte LDL-R in normocholesterolemic condition, also produced no effects on plasma lipid parameters. LDL binding studies using macrophages isolated from transplanted mice showed a lack of LDL-R expression. Thus, despite their large number and wide distribution, bone marrow-derived cells do not significantly influence receptor-mediated clearance of plasma LDL.

Asunto(s)

Leucocitos/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores de LDL/genética , Animales , Secuencia de Bases , Trasplante de Médula Ósea/métodos , Trasplante de Médula Ósea/fisiología , Células Cultivadas , Colesterol/sangre , Colesterol/metabolismo , Cromatografía en Agarosa , ADN/análisis , ADN/genética , Cartilla de ADN/química , Electroforesis en Gel de Agar , Citometría de Flujo , Técnicas de Transferencia de Gen , Radioisótopos de Yodo , Leucocitos/citología , Leucocitos/fisiología , Lipoproteínas/análisis , Lipoproteínas/clasificación , Lipoproteínas LDL/análisis , Lipoproteínas LDL/sangre , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Receptores de LDL/química , Receptores de LDL/metabolismo , Factores de Tiempo

RESUMEN

Highly purified human blood burst-forming units-erythroid (BFU-E) were used to study the effects of interferon gamma (IFN gamma). IFN gamma inhibited erythroid colony formation, cell proliferation, and differentiation of day 3 to day 6 mature BFU-E in a dose-dependent manner. The primitive BFU-E (day 1 and day 2 cells) and later day 7 cells were less affected. IFN gamma dose-response experiments demonstrated that the number and size of erythroid colonies were reduced at a concentration of 500 U/ml with more complete inhibition at 1,000 U/ml. Inhibition of day 4 to day 6 erythroid progenitors was first noted by 72 h of incubation with IFN gamma, and target cell growth and differentiation continued to decrease with further incubation. IFN gamma also induced erythroblast apoptosis which was demonstrated by both nuclear condensation and fragmentation plus flow cytometry with in situ end-labelling. Because day 3 to day 6 cells need stem cell factor (SCF) for development in serum-free culture, the relationship of IFN gamma inhibition to this growth factor was investigated. The reduction in the number of erythroid colonies by IFN gamma was reversed by SCF although the colony size was not completely re-established. In contrast, interleukin-3 did not have the capacity to overcome the inhibitory effects of IFN gamma. Since IFN gamma blood levels are elevated in some anemias of chronic disease, IFN gamma may have a role in promoting this anemia and its inhibitory effect might be better overcome by SCF plus EP. However, the mechanism by which these growth factors overcome the inhibition of IFN gamma, or vice versa, is unknown at the present time.

Asunto(s)

Células Precursoras Eritroides/efectos de los fármacos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/farmacología , Factor de Células Madre/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Senescencia Celular , Eritroblastos/efectos de los fármacos , Humanos , Factores de Tiempo

RESUMEN

Two peaks of methionine adenosyltransferase (MAT) activity from human erythrocytes were partially purified on a DEAE-cellulose column. Using anti-MAT antibodies, a 60 kDa form of MAT, referred to as rho was identified in peak I. Although rho represented the major MAT protein in crude erythrocyte extracts, the enzyme was very labile and accounted for only 6% of the total MAT activity. Peak II enzyme was stable, and consisted of the previously described catalytic alpha (53 kDa) subunit and the beta subunit (38 kDa), both of which are found in activated human lymphocytes and leukemic cells of lymphoid origin. Mature normal and polycythemic erythrocytes contained predominantly rho as the major MAT protein, while nucleated erythrocytes and reticulocytes contained predominantly the lambda (68 kDa), the major form found in resting human lymphocytes. Human erythroleukemic cells (HEL 92.1.7) contained the alpha, alpha' and beta subunits of MAT, and in this regard was indistinguishable from MAT found in activated lymphocytes and leukemic cells of lymphoid origin (Jurkat). Since rho was generated during the incubation of extracts from resting lymphocytes, which contain predominantly lambda, in the absence of protease inhibitors; the rho form of MAT appears to be derived from the lambda form by proteolytic cleavage. The data indicate that distinct forms of MAT are present at different stages of erythrocyte maturation and reveal the presence of a new form of MAT with reduced activity compared to previously described forms.

Asunto(s)

Eritrocitos/enzimología , Leucemia Eritroblástica Aguda/enzimología , Metionina Adenosiltransferasa/química , Separación Celular , Sangre Fetal , Humanos , Metionina Adenosiltransferasa/aislamiento & purificación

Asunto(s)

Células Precursoras Eritroides/citología , Hemoglobina Fetal/análisis , Hibridación Fluorescente in Situ/métodos , Embarazo/sangre , Sondas de ADN , Femenino , Humanos , Análisis para Determinación del Sexo , Cromosoma X , Cromosoma Y

RESUMEN

This study assessed validity and reliability of the Health Knowledge Inventory-Alpha (HKI-Alpha) in a sample of high school seniors. The HKI-Alpha, a general health knowledge test, consists of 110 multiple choice items covering 11 health content areas. All seniors attending one of four high schools completed HKI-Alpha twice, one week apart. A secondary sample of college students also was tested. Descriptive analysis revealed the test discriminated among examinees and avoided ceiling and floor effects. Test-retest reliability was .81 (n = 355). Internal consistency reliability (KR20) was .85 (n = 418). A sample of college students scored significantly higher than the high school students, demonstrating construct validity. Other estimates of content validity, criterion-related validity, and construct validity were high.

Asunto(s)

Pruebas de Aptitud , Conocimientos, Actitudes y Práctica en Salud , Servicios de Salud Escolar , Adolescente , Humanos , Reproducibilidad de los Resultados

RESUMEN

A long-sought goal of medical genetics has been development of prenatal diagnostic procedures that do not endanger the conceptus. Reliable and universal screening for cytogenetic disorders would require analysis of fetal cells isolated from the maternal circulation. This would be applicable to all pregnant women, irrespective of their ages or histories. In the current study fetal nucleated erythrocytes were flow sorted on the basis of four parameters: cell size, cell granularity, transferrin receptor, and glycophorin-A cell surface molecule. By polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific deoxyribonucleic acid sequences, male fetuses were correctly identified among flow-sorted samples in 12 of 12 (100%) pregnancies; female fetuses were correctly identified in 5 of 6 (83%) pregnancies. We also achieved the prenatal diagnosis of fetal aneuploidies by use of flow-sorted nucleated fetal erythrocytes and in situ hybridization with chromosome-specific deoxyribonucleic acid probes: one case of trisomy 21 that was detected in maternal blood taken 1 week after chorionic villus sampling and one case of trisomy 18 that was detected in maternal blood taken immediately before chorionic villus sampling. Although our results are promising, additional data on the background sensitivity and specificity of in situ hybridization in flow-sorted fetal cells will be necessary to minimize subjective interpretation and permit clinical application.

Asunto(s)

Cromosomas Humanos Par 18 , Eritrocitos/patología , Pruebas Genéticas/métodos , Análisis para Determinación del Sexo , Trisomía , Adulto , Aneuploidia , Femenino , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa , Embarazo

RESUMEN

Anti-melanoma monoclonal antibody XMMME-001 was cross-linked to anti-CD3 monoclonal antibody OKT3 with succinimidyl 3(2-pyridyldithio)propionate (SPDP), and 2-iminothiolane. The dimer heteroconjugate was purified by HPLC gel filtration, labeled with 131I, and 10 micrograms was injected into each of 24 BALB/c mice. The dimeric heteroconjugate's blood survival in sequentially bled mice (n = 3) and its biodistribution in organs of sacrificed mice (n = 21) were studied. In plasma, the heteroconjugate showed an alpha phase with a half-life of 4 h, and a beta phase with a half-life of about 18 h. Electrophoretic analysis of labeled heteroconjugate in plasma showed that the half-life of disulfide bonds linking the monoclonal antibodies was approximately 7-8 h. Radioactive heteroconjugate accumulated primarily in the liver; significant uptake was also seen in white blood cells and spleen. Very little radioactivity accumulated in kidney, lung, or colon. As a comparison, SPDP-derivatized anti-melanoma antibody was studied by the same methods. It showed an average alpha-phase half-life of 12.5 h; its maximum accumulation in liver or white blood cells was less than 30% of that of the heteroconjugate. Very low levels accumulated in other tissues. The results imply that the shorter half-life in plasma of the heteroconjugate is primarily determined by clearance due to its larger size and conformation, not the lability of cross-linking disulfide bonds.

Asunto(s)

Anticuerpos Monoclonales/análisis , Inmunotoxinas/farmacocinética , Melanoma/inmunología , Animales , Anticuerpos Antineoplásicos/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3 , Reactivos de Enlaces Cruzados/farmacología , Disulfuros/sangre , Estabilidad de Medicamentos , Femenino , Semivida , Inmunotoxinas/sangre , Ratones , Ratones Endogámicos BALB C , Plasma , Receptores de Antígenos de Linfocitos T/inmunología , Succinimidas/farmacología , Distribución Tisular

RESUMEN

OKT3 and BABR1 [anti-(breast tumour) antibody] were conjugated using N-succinimidyl-3-(2-pyridy]dithio)propionate (SPDP). The procedure employed mild reducing conditions for SPDP-BABR1 and short conjugation incubations at 37 degrees C. The bifunctional immunoconjugates thus produced were isolated by HPLC gel filtration on a preparative TSK 3000 SW column. Both intact IgG and F(ab')2 fragments have been conjugated. The ratio of SPDP to IgG for the optimal yield of dimeric OKT3-BABR1 heteroconjugates was determined to be 2 for OKT3 and 1-2 for BABR1. The OKT3-BABR1 dimers were shown to be bifunctional heteroconjugates by polyacrylamide gel electrophoresis, isoelectric focusing, radial immunodiffusion, and flow cytometry. The binding specificities of the bifunctional heteroconjugates were identical to the specificities of both parent antibodies.

Asunto(s)

Anticuerpos Monoclonales/análisis , Anticuerpos Antineoplásicos/análisis , Neoplasias de la Mama/inmunología , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Humanos , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G/análisis , Focalización Isoeléctrica