RESUMEN
Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the dystrophin gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.
Asunto(s)
Distrofina/deficiencia , Serotonina/metabolismo , Pez Cebra/metabolismo , Animales , Birrefringencia , Evaluación Preclínica de Medicamentos , Distrofina/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Receptores de Serotonina , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Análisis de SupervivenciaRESUMEN
BACKGROUND: It has long been recognized that bipolar disorder (BD) and attention deficit-hyperactivity disorder (ADHD) co-occur in an uncertain proportion of patients, recognized commonly in juvenile years. There is growing suspicion that such co-occurrence is associated with several clinically unfavorable characteristics. Accordingly, we compared 703 type I or II BD subjects with vs. without a lifetime diagnosis of ADHD. METHODS: We compared 173 BD patients with vs. 530 without co-occurring ADHD for selected demographic and clinical factors, using standard initial bivariate comparisons followed by multivariable logistic regression modeling. RESULTS: ADHD was found in 25% of BD subjects, more among men and with type I BD. Those with ADHD had higher scores at the Adult ADHD Self-Report Scale (ASRS), were more likely to have had less successful school performance, unemployment, lower socioeconomic status, less marriage and more divorce, as well as more substance abuse, suicide attempts, and [hypo]mania, but were less likely to have an anxiety disorder or a family history of mood disorder. Multivariable logistic regression modeling found six factors differing between BD subjects with versus without ADHD: less education after high school, higher ASRS score for inattention, ever separated or divorced, irritable temperament, male sex, and lower scores on the Hamilton Depression Rating Scale (HDRS) at intake. COMMENTS: Co-occurrence of ADHD with BD was identified at a moderate rate, and was associated with several unfavorable outcomes as well as a tendency toward [hypo]mania.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/epidemiología , Trastorno Bipolar/epidemiología , Adulto , Trastorno por Déficit de Atención con Hiperactividad/psicología , Trastorno Bipolar/psicología , Comorbilidad , Femenino , Humanos , Masculino , TemperamentoRESUMEN
Affective temperament has been suggested as a potential mediator of the effect between genetic predisposition and neurocognitive functioning. As such, this report seeks to assess the extent of the correlation between affective temperament and cognitive function in a group of bipolar II subjects. 46 bipolar II outpatients [mean age 41.4 years (SD 18.2); female 58.9%] and 46 healthy controls [mean age 35.1 years (SD 18); female 56.5%] were evaluated with regard to their demographic and clinical characteristics, affective temperament, and neurocognitive performance. Crude bivariate correlation analyses and multiple linear regression models were constructed between five affective temperament subscales and eight neurocognitive domains. Significant correlations were identified in bipolar patients between hyperthymic temperament and verbal memory and premorbid IQ; cyclothymic temperament and attention; and irritable temperament, attention, and verbal fluency. In adjusting for potential confounders of the relationship between temperament and cognitive function, the strongest mediating factors among the euthymic bipolar patients were found to be residual manic and depressive symptoms. It is therefore concluded that affective temperaments may partially influence the neurocognitive performance of both healthy controls and euthymic patients with bipolar disorder type II in several specific domains.
Asunto(s)
Síntomas Afectivos/psicología , Trastorno Bipolar/psicología , Cognición , Trastorno Ciclotímico/psicología , Temperamento , Adulto , Anciano , Atención , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Genio Irritable , Modelos Lineales , Masculino , Persona de Mediana EdadRESUMEN
In recent years, investigators have begun to consider the possibility of explaining the physiopathology of bipolar disorder from a neuroprogressive perspective. The evidence that supports the feasibility of such an approach is varied, and arises from neuroimaging studies, batteries of neurocognitive evaluations, and tests to identify the specific biomarkers of the disorder. The present article seeks to perform a review of the research that investigates the cognitive deficits in bipolar disorder. A bibliographic revision was performed of articles published between 1990 and 2015. Levels of cognitive performance were explored in both cross-sectional and longitudinal studies. The compiled studies signal the presence of altered cognitive function, even during periods of euthymia. However, there are contradictory results as to whether bipolar disorder presents a degenerative course. New lines of investigation suggest that only a percentage of individuals with bipolar disorder are affected in a progressive manner. It is of paramount importance to perform new longitudinal studies in high-risk populations, so as to validate or refute a neuroprogressive model of cognitive deficits in patients with bipolar disorder.
Asunto(s)
Trastorno Bipolar/complicaciones , Trastorno Bipolar/psicología , Trastornos del Conocimiento/etiología , Cognición , Progresión de la Enfermedad , HumanosRESUMEN
More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations.
Asunto(s)
Ácido Ascórbico/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/farmacología , Línea Celular Tumoral , Ácido Deshidroascórbico/metabolismo , Femenino , Transportador de Glucosa de Tipo 1/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Ratones , Ratones Mutantes , Ratones Desnudos , Proteínas Proto-Oncogénicas p21(ras)/genética , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.
Asunto(s)
Acetaldehído/farmacología , Resistencia a Antineoplásicos/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Animales , Western Blotting , Línea Celular Tumoral , Anemia de Fanconi/genética , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.
Asunto(s)
Metilación de ADN , Heterocigoto , Isocitrato Deshidrogenasa/genética , Mutación , Alelos , Western Blotting , Inmunoprecipitación de Cromatina/métodos , Regulación hacia Abajo , Epigenómica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Silenciador del Gen , Sitios Genéticos , Células HCT116 , Histonas/genética , Histonas/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/genética , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Phosphotidylinositol-3-kinase (PI3K) signaling is altered in the majority of human cancers. To gain insight into the roles of members of this pathway in growth regulation, we inactivated AKT1, AKT2, or PDPK1 genes by targeted homologous recombination in human colon cancer cell lines. Knockout of either AKT1 or AKT2 had minimum effects on cell growth or downstream signaling. In contrast, knockout of both AKT1 and AKT2 resulted in markedly reduced proliferation in vitro when growth factors were limiting and severely affected experimental metastasis in mice. Unexpectedly, AKT1 and AKT2 appeared to regulate growth through FOXO proteins, but not through either GSK3beta or mTOR. In contrast, inactivation of PDPK1 affected GSK3beta and mTOR activation. These findings show that the PI3K signaling pathway is wired differently in human cancer cells than in other cell types or organisms, which has important implications for the design and testing of drugs that target this pathway.
Asunto(s)
Proliferación Celular , Neoplasias Colorrectales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Apoptosis , Western Blotting , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HCT116 , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR , Trasplante HeterólogoRESUMEN
Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently up-regulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low-glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low-glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired KRAS mutations not present in their parents. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.
Asunto(s)
Neoplasias Colorrectales/genética , Genes ras , Glucosa/metabolismo , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Marcación de Gen , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Ácido Láctico/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Piruvatos/farmacología , Trasplante HeterólogoRESUMEN
Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenous TP53 status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent up-regulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G(2)/M transition in the cells whose TP53 genes were inactivated compared with those with WT TP53 genes. This led to the hypothesis that the viability of stressed cells without WT TP53 depended on PLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WT TP53 alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.
Asunto(s)
Neoplasias/patología , Neoplasias/terapia , Proteína p53 Supresora de Tumor/metabolismo , Alelos , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1/efectos de los fármacos , Perfilación de la Expresión Génica , Marcación de Gen , Genotipo , Humanos , Imidazoles/metabolismo , Ratones , Piperazinas/metabolismo , Pteridinas/farmacología , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Deltaexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2(Deltaex11/Deltaex11) and BRCA2(Deltaex11/Y3308X) were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2(Deltaex11/Y3308Y), BRCA2(Deltaex11/P3292L), and BRCA2(Deltaex11/P3280H) had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2(Deltaex11/S3291E)) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2(Deltaex11/S3291A)) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.
Asunto(s)
Biblioteca de Genes , Genes BRCA2 , Variación Genética/fisiología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inestabilidad Cromosómica/efectos de los fármacos , Inestabilidad Cromosómica/efectos de la radiación , Células Clonales , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Etopósido/farmacología , Humanos , Mitomicina/farmacología , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Ensayo de Tumor de Célula MadreRESUMEN
Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.
Asunto(s)
Técnicas de Cultivo de Célula , Marcación de Gen/métodos , Ingeniería Genética/métodos , Alelos , Línea Celular , Células Cultivadas , Dependovirus/genética , Vectores Genéticos , Humanos , Recombinación GenéticaRESUMEN
Internal human xenografts provide valuable animal models to study the microenvironments and metastatic processes occurring in human cancers. However, the use of such models is hampered by the logistical difficulties of reproducibly and simply assessing tumor burden. We developed a high-sensitivity assay for quantifying human DNA in small volumes of mouse plasma, enabling in-life monitoring of systemic tumor burden. Growth kinetics analyses of various xenograft models showed the utility of circulating human DNA as a biomarker. We found that human DNA concentration reproducibly increased with disease progression and decreased after successful therapeutic intervention. A marked, transient spike in circulating human tumor DNA occurred immediately after cytotoxic therapy or surgery. This simple assay may find broad utility in target validation studies and preclinical drug development programs.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias del Colon/sangre , ADN de Neoplasias/sangre , Animales , Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Cartilla de ADN , Modelos Animales de Enfermedad , Femenino , Células HCT116 , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
BACKGROUND & AIMS: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. CONCLUSIONS: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.
Asunto(s)
Adenocarcinoma/genética , Rotura Cromosómica/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Adenocarcinoma/tratamiento farmacológico , Alelos , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína del Grupo de Complementación C de la Anemia de Fanconi/efectos de los fármacos , Proteína del Grupo de Complementación G de la Anemia de Fanconi/efectos de los fármacos , Humanos , Inmunoprecipitación , Hibridación in Situ , Cariotipificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that directly transactivates genes important for the growth and metabolism of solid tumors. HIF-1alpha is overexpressed in cancer, and its level of expression is correlated with patient mortality. Increased synthesis or stability of HIF-1alpha can be induced by hypoxia-dependent or hypoxia-independent factors. Thus, HIF-1alpha is expressed in both nonhypoxic and hypoxic cancer cells. The role of HIF-1alpha in nonhypoxia-mediated cancer cell proliferation remains speculative. We have disrupted HIF-1alpha by targeted homologous recombination in HCT116 and RKO human colon cancer cells. Loss of HIF-1alpha significantly reduced nonhypoxia-mediated cell proliferation in vitro and in vivo. Paradoxically, loss of HIF-1alpha expression did not grossly affect the hypoxic compartments within tumor xenografts in vivo, although HIF-1alpha promoted cell proliferation and survival under hypoxia in vitro. To further test the role of HIF-1alpha within tumor compartments, we generated cells with combined disruptions of both HIF-1alpha and vascular endothelial growth factor (VEGF). In all xenografts, disruption of VEGF led to marked expansion of the hypoxic compartments and growth delay. Nonetheless, the presence or absence of HIF-1alpha did not grossly affect these expanded hypoxic compartments. These data provide compelling evidence that, in a subset of colon cancers, (a) HIF-1alpha is a positive factor for nonhypoxia-mediated cell proliferation in vitro and in vivo and (b) HIF-1alpha is a positive factor for cell proliferation and survival under hypoxic conditions in vitro, but does not grossly contribute to the tumor hypoxic compartments in vivo.
Asunto(s)
Neoplasias del Colon/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Animales , Procesos de Crecimiento Celular/genética , Procesos de Crecimiento Celular/fisiología , Hipoxia de la Célula , Supervivencia Celular/genética , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , ADN de Neoplasias/biosíntesis , Femenino , Glucólisis , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Desnudos , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
GSTP1 is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles with glutathione in the process of detoxification. GSTP1 is widely overexpressed in colorectal cancer, from aberrant crypt foci to advanced carcinomas. Increased expression of GSTP1 is associated with multidrug resistance and a worse clinical prognosis. However, GSTP1-null mice have an increased risk of tumor formation. Thus, the biological function of GSTP1 in colorectal cancer biology remains speculative. In an effort to gain further insights into the role of GSTP1 in tumorigenesis, we disrupted the GSTP1 gene in HCT116 human colorectal cancer cells using targeted homologous recombination. We find that loss of GSTP1 resulted in impaired clonogenic survival and proliferation. Specifically, under growth-limiting conditions, (a) GSTP1 protected HCT116 cells from oxidative stress and associated apoptosis and (b) promoted mitogen-activated protein kinase-extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase-mediated G1-S cell cycle progression. In vivo, GSTP1 was critical for engraftment and growth of HCT116 tumor xenografts. These studies directly show that GSTP1 promotes clonogenic survival and proliferation in HCT116 human colon cancer cells.
Asunto(s)
Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Gutatión-S-Transferasa pi/genética , Animales , Apoptosis/fisiología , Procesos de Crecimiento Celular/genética , Neoplasias del Colon/genética , Femenino , Fase G1/fisiología , Gutatión-S-Transferasa pi/biosíntesis , Gutatión-S-Transferasa pi/metabolismo , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estrés Oxidativo , Recombinación Genética , Fase S/fisiologíaRESUMEN
PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.
Asunto(s)
Proliferación Celular , Invasividad Neoplásica/patología , Fosfatidilinositol 3-Quinasas/genética , Sustitución de Aminoácidos , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/genética , Cromonas/farmacología , Fosfatidilinositol 3-Quinasa Clase I , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead , Marcación de Gen , Sustancias de Crecimiento/deficiencia , Humanos , Insulina/deficiencia , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Desnudos , Morfolinas/farmacología , Mutación , Invasividad Neoplásica/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Cell-specific gene expression profiling from heterogeneous human tissues is confounded by cell purification limitations. Here, we describe a technique to generate gene expression profiles of pure populations of prostate cancer cells obtained from fresh-frozen prostatectomy specimens and small initial quantities of RNA by combining laser capture microdissection and microserial analysis of gene expression (LCM-microSAGE). Two microSAGE libraries were obtained from approximately 100,000 laser pulses, estimated to contain fewer than 3 x 10(5) cells and 20-30 ng mRNA. Two libraries were sequenced to a depth of 10,111 and 10,463 unique tags from normal and cancer cells, representing 6453 and 6923 genes, respectively. Most transcripts were expressed at similar levels, but cancer cells compared with normal cells had increased expression of 385 tags and decreased expression of 389 tags. A total of 20 genes were differentially expressed (P<0.05); five of these genes were upregulated and 15 were downregulated in cancer cells. Quantitative reverse transcriptase-polymerase chain reaction results from three selected genes corroborated the existence of cell-specific gene expression in LCM-microSAGE-derived libraries. In conclusion, the LCM-microSAGE approach demonstrates that large-scale expression profiles of known and unknown transcripts can be generated from pure populations of target cells obtained from human tissue samples comprised of heterogeneous mixtures of cell types.
Asunto(s)
Perfilación de la Expresión Génica , Microdisección/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias de la Próstata/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Rayos Láser , Masculino , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Reproducibilidad de los ResultadosRESUMEN
Although the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to play an important role in the immunosurveillance of neoplasia, apoptotic factors that modulate the sensitivity of cancer cells to TRAIL are poorly understood. The inhibitor of apoptosis proteins (IAPs) have generated considerable interest as potential targets for cancer therapy, but the lack of a phenotype in X-linked IAP (XIAP) knockout mice has generated speculation that IAP function may be redundant. Using gene targeting technology, we show that disruption of the gene encoding XIAP in human cancer cells did not interfere with basal proliferation, but caused a remarkable sensitivity to TRAIL. These results demonstrate that XIAP is a nonredundant modulator of TRAIL-mediated apoptosis and provide a rationale for XIAP as a therapeutic target.