RESUMEN
Boron neutron capture therapy (BNCT) is a re-emerging binary cellular level cancer intervention that occurs through the interaction of a cancer-specific 10boron (10B) drug and neutrons. We created a new 10B drug, 3-borono-l-tyrosine (BTS), that improves on the characteristics of the main historical BNCT drug 4-borono-l-phenylalanine (BPA). BTS has up to 4 times greater uptake in vitro than BPA and increased cellular retention. Like BPA, BTS uptake is mediated by the l-type amino acid transporter-1 (LAT1) but is less sensitive to natural amino acid competition. BTS can be formulated and bolus dosed at much higher levels than BPA, resulting in 2-3 times greater boron delivery in vivo. Fast blood clearance and greater tumor boron delivery result in superior tumor-to-blood ratios. BTS boron delivery appears to correlate with LAT1 expression. BTS is a promising boron delivery drug that has the potential to improve modern BNCT interventions.
Asunto(s)
Aminoácidos , Terapia por Captura de Neutrón de Boro , Línea Celular Tumoral , Boro , Terapia por Captura de Neutrón de Boro/métodos , Solubilidad , Fenilalanina/química , Compuestos de Boro/químicaRESUMEN
High mannose (HM) glycan levels on secreted monoclonal antibodies can be influenced by external factors, including osmolality and copper deficiency, and by intrinsic factors determined by different cell lines. In order to identify the metabolic markers associated with HM glycan levels, metabolomics analysis was performed to assess the changes in the extracellular metabolites of recombinant cell lines at different time points during fed-batch production process. Ornithine was identified as the common metabolic marker influenced by both external and intrinsic factors when eight different medium conditions and eight different cell lines exhibiting different levels of HM were compared. A strong correlation was also observed between HM and mRNA expression levels of arginase 1, an enzyme that catalyzes the conversion of arginine to ornithine. The results from functional validation study showed that the supplementation of ornithine to the culture medium leads to an increased level of HM, while reduced concentration of spermine, a downstream product of ornithine metabolism, leads to a decreased level of HM. Additional metabolic markers correlating with HM glycan levels were identified from eight-cell line comparison analysis. A common feature shared by these identified markers is their previously described roles as contributors of cellular redox regulation.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Manosa/metabolismo , Polisacáridos/metabolismo , Animales , Arginasa/genética , Células CHO , Cobre/metabolismo , Cricetinae , Cricetulus , Medios de Cultivo , Metabolómica , Ornitina/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Espermina/metabolismoRESUMEN
Insufficient copper level in the mammalian cell culture medium resulted in lactate accumulation while maintaining similar growth and culture viability profiles. Label-free, LC-MS/MS-based shotgun proteomics method was applied to compare the protein expression profiles obtained from the cultures exposed to suboptimal copper level to those provided with sufficient amount of copper. Under copper deficient condition, a substantial reduction of the protein levels of the multiple subunits of Complex IV, also known as cytochrome c oxidase, of the mitochondrial electron transport chain was observed for all three different Chinese Hamster Ovary (CHO) cell lines expressing therapeutic monoclonal antibodies tested. Additional proteins affected by suboptimal copper level included peroxiredoxin (PRDX) and hepatocyte-derived growth factor (HDGF), which were affected during early phase of the fed-batch production, several days prior to initiation of lactate accumulation. In contrast, proteins such as syntenin (SDCBP) and integral membrane 2C (ITM2C) showed altered expression patterns toward the end of culture duration, after lactate divergence had occurred. For all conditions tested, time was the most predominant factor facilitating the direction of global protein expression trend, with substantial number of proteins subjected to time-dependent changes in expression, independent of copper.
Asunto(s)
Cobre/deficiencia , Cobre/metabolismo , Proteómica/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Complejo IV de Transporte de Electrones/metabolismo , Ácido Láctico/metabolismoRESUMEN
Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest that productivity is rather a minor feature in the context of global transcriptional or protein expression space.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/metabolismo , Proteómica/métodos , Biología de Sistemas/métodos , Animales , Anticuerpos Monoclonales/genética , Células CHO , Línea Celular , Proliferación Celular , Análisis por Conglomerados , Cricetinae , Cricetulus , Perfilación de la Expresión Génica , Análisis de Componente Principal , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Adequate supply of nutrients, especially providing a sufficient level of specific amino acids, is essential for cell survival and production. Complex raw materials such as soy hydrolysates or yeast extracts are the source for both free amino acids and peptides. However, typical chemically defined (CD) media provide amino acids only in free form. While most amino acids are highly soluble in media and can be provided at fairly high concentrations, certain amino acids such as tyrosine have poor solubility and thus, only a limited amount can be added as a media component. The limited solubility of amino acids in media can raise the risk of media precipitation and instability, and could contribute to suboptimal culture performance due to insufficient nutrient levels to meet cellular demands. In this study, we examine the use of chemically synthesized dipeptides as an alternative method for delivering amino acids to various monoclonal antibody producing cell lines. In particular, we focus on tyrosine-containing dipeptides. Due to their substantially higher solubility (up to 250-fold as compared with free tyrosine), tyrosine-containing dipeptides can efficiently provide large amounts of tyrosine to cultured cells. When tested in fed-batch processes, these supplemental dipeptides exerted positive effects, including enhanced culture viability and titer. Moreover, dipeptide-supplemented cultures displayed improved metabolic profiles including lower lactate and NH 4(+) production, and better pH maintenance. In bioreactor studies using two-sided pH control, a lactate spike occurring on Day 10 and the concomitant high levels of base addition could be prevented with dipeptide supplementation. These beneficial effects could be obtained by one-time addition of dipeptides during inoculation, and did not require further feeds during the entire 11-15-day process. Non-tyrosine-containing dipeptides, such as His-Gly, also showed improved productivity and viability over control cultures.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Histidina/metabolismo , Tirosina/metabolismo , Animales , Reactores Biológicos , Biotecnología , Células CHO , Supervivencia Celular/fisiología , Cricetinae , Cricetulus , Medios de Cultivo/química , Histidina/química , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Lisina/química , Lisina/metabolismo , Tirosina/químicaRESUMEN
The catalytic and regulatory subunits of class I phosphoinositide 3-kinase (PI3K) have oncogenic potential. The catalytic subunit p110α and the regulatory subunit p85 undergo cancer-specific gain-of-function mutations that lead to enhanced enzymatic activity, ability to signal constitutively, and oncogenicity. The ß, γ, and δ isoforms of p110 are cell-transforming as overexpressed wild-type proteins. Class I PI3Ks have the unique ability to generate phosphoinositide 3,4,5 trisphosphate (PIP(3)). Class II and class III PI3Ks lack this ability. Genetic and cell biological evidence suggests that PIP(3) is essential for PI3K-mediated oncogenicity, explaining why class II and class III enzymes have not been linked to cancer. Mutational analysis reveals the existence of at least two distinct molecular mechanisms for the gain of function seen with cancer-specific mutations in p110α; one causing independence from upstream receptor tyrosine kinases, the other inducing independence from Ras. An essential component of the oncogenic signal that is initiated by PI3K is the TOR (target of rapamycin) kinase. TOR is an integrator of growth and of metabolic inputs. In complex with the raptor protein (TORC1), it controls cap-dependent translation, and this function is essential for PI3K-initiated oncogenesis.
Asunto(s)
Neoplasias/etiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Transformación Celular Neoplásica , Humanos , Isoenzimas/fisiología , Mutación , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
Phosphatidylinositol 3-kinases (PI3K) are divided into three classes, which differ in their substrates and products. Class I generates the inositol phospholipids PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 referred as PIP, PIP2, and PIP3, respectively. Class II produces PIP and PIP2, and class III generates only PIP. Substrate and product differences of the three classes are determined by the activation loops of their catalytic domains. Substitution of the class I activation loop with either class II or III activation loop results in a corresponding change of substrate preference and product restriction. We have evaluated such activation loop substitutions to show that oncogenic activity of class I PI3K is linked to the ability to produce PIP3. We further show that reduction of cellular PIP3 levels by the 5'-phosphatase PIPP interferes with PI3K-induced oncogenic transformation. PIPP also attenuates signaling through Akt and target of rapamycin. Class III PI3K fails to induce oncogenic transformation. Likewise, a constitutively membrane-bound class I PI3K mutant retaining only the protein kinase is unable to induce transformation. We conclude that PIP3 is an essential component of PI3K-mediated oncogenesis and that inability to generate PIP3 abolishes oncogenic potential.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Dominio Catalítico , Línea Celular , Embrión de Pollo , Fibroblastos/metabolismo , Inositol Polifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Cancer-specific mutations in the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) p110 alpha occur in diverse tumors in frequencies that can exceed 30%. The majority of these mutations map to one of three hot spots in the gene, and the rest are distributed over much of the PI3K coding sequence. Most of the cancer-specific mutations induce a gain of function that results in oncogenicity, elevated lipid kinase activity and constitutive signaling through the kinases Akt and TOR. The location of the mutations on a model structure of p110 alpha indicates several distinct mechanisms for the gain of function. The mutated p110 alpha proteins are promising cancer targets. Although identification of mutant-specific small-molecule inhibitors seems technically challenging, the therapeutic benefits from such inhibitors could be extremely important.
Asunto(s)
Mutación/genética , Neoplasias/enzimología , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Humanos , Isoenzimas/metabolismo , Modelos Biológicos , Neoplasias/terapia , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/clasificaciónRESUMEN
Unlike the livers of humans and mice, and most hepatoma cells, which accumulate triglycerides when treated with microsomal triglyceride transfer protein (MTP) inhibitors, L35 rat hepatoma cells do not express MTP and cannot secrete very low density lipoprotein (VLDL), yet they do not accumulate triglyceride. In these studies we show that transcriptional co-repression of the two lipid transfer proteins, liver fatty acid-binding protein (L-FABP) and MTP, which cooperatively shunt fatty acids into de novo synthesized glycerolipids and the transfer of lipids into VLDL, respectively, act together to maintain hepatic lipid homeostasis. FAO rat hepatoma cells express L-FABP and MTP and demonstrate the ability to assemble and secrete VLDL. In contrast, L35 cells, derived as a single cell clone from FAO cells, do not express L-FABP or MTP nor do they assemble and secrete VLDL. We used these hepatoma cells to elucidate how a conserved DR1 promoter element present in the promoters of L-FABP and MTP affects transcription, expression, and VLDL production. In FAO cells, the DR1 elements of both L-FABP and MTP promoters are occupied by peroxisome proliferator-activated receptor alpha-retinoid X receptor alpha (RXRalpha), with which PGC-1beta activates transcription. In contrast, in L35 cells the DR1 elements of both L-FABP and MTP promoters are occupied by chicken ovalbumin upstream promoter transcription factor II, and transcription is diminished. The combined findings indicate that peroxisome proliferator-activated receptor alpha-RXRalpha and PGC-1beta coordinately up-regulate L-FABP and MTP expression, by competing with chicken ovalbumin upstream promoter transcription factor II for the DR1 sites in the proximal promoters of each gene. Additional studies show that ablation of L-FABP prevents hepatic steatosis caused by treating mice with an MTP inhibitor. Our findings show that reducing both L-FABP and MTP is an effective means to reduce VLDL secretion without causing hepatic steatosis.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión a Ácidos Grasos/genética , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Transcripción Genética/genética , Animales , Apolipoproteínas B/metabolismo , Secuencia de Bases , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Línea Celular , Dimerización , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/metabolismo , Hígado Graso/genética , Hígado Graso/metabolismo , Expresión Génica/genética , Genes Reporteros/genética , Factor Nuclear 1-beta del Hepatocito/metabolismo , Masculino , Ratones , Ratones Noqueados , PPAR alfa/agonistas , PPAR alfa/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , Ratas , Receptores X Retinoide/agonistas , Receptores X Retinoide/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The potential oncogenicity of PI 3-kinases is revealed by two principal mechanisms: mutations causing gain of function and overexpression of wild-type proteins. Cancer-specific mutations in PIK3CA, the gene coding for the catalytic subunit p110alpha of PI 3-kinase, are oncogenic in the animal. These mutations are therefore significant determinants of the oncogenic cellular phenotype in human tumors and are appropriate and promising targets for small molecule inhibitors. Overexpression of wild-type p110beta, gamma and delta induces oncogenic transformation in cell culture. Although these non-alpha isoforms of PI 3-kinase have not been found mutated in human cancer, deregulated expression could contribute to cellular oncogenic properties and deserves increased attention.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/genética , Animales , Transformación Celular Neoplásica/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
In this issue of Cancer Cell, Fan and coworkers describe a novel inhibitor of PI3 kinase (PI3K) that potently interferes with the growth of glioma cells. They show that the efficacy of this inhibitor results from dual, synergistic activity against the p110alpha subunit of PI3K and against TOR. Although p110alpha and TOR belong to the same signaling pathway, they both must be inactivated because of the need to silence the regulatory feedback loop that remains unaffected by monospecific inhibitors. The new PI3K inhibitor achieves the effects of combination therapy as a single agent by fortuitously hitting two critical targets.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Animales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/uso terapéutico , Especificidad por SustratoRESUMEN
The PIK3CA gene, coding for the catalytic subunit p110alpha of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110alpha proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110alpha, E542K, E545K, and H1047R, are oncogenic in vivo. They induce tumors in the chorioallantoic membrane of the chicken embryo and cause hemangiosarcomas in the animal. These tumors are marked by increased angiogenesis and an activation of the Akt pathway. The target of rapamycin inhibitor RAD001 blocks tumor growth induced by the H1047R p110alpha mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.
Asunto(s)
Mutación , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Animales , Antineoplásicos/farmacología , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Pollos , Membrana Corioalantoides/metabolismo , Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Hemangiosarcoma/metabolismo , Humanos , Microglía/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Placebos , Transducción de Señal , Sirolimus/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Class I phosphoinositide 3-kinase contains four isoforms of the catalytic subunit, p110alpha, -beta, -gamma, and -delta. At physiological levels of expression, the wild-type p110alpha isoform lacks oncogenic potential, but gain-of-function mutations and overexpression of p110alpha are correlated with oncogenicity. The p110beta, -gamma, and -delta isoforms induce transformation of cultured cells as wild-type proteins. This oncogenic potential requires kinase activity and can be suppressed by the target of rapamycin inhibitor rapamycin. The p110delta isoform constitutively activates the Akt signaling pathway; p110gamma activates Akt only in the presence of serum. The isoforms differ in their requirements for upstream signaling. The transforming activity of the p110gamma isoform depends on rat sarcoma viral oncogene homolog (Ras) binding; preliminary data suggest the same for p110beta and indicate Ras-independent oncogenic potential of p110delta. The surprising oncogenic potential of the wild-type non-alpha isoforms of class I phosphoinositide 3-kinase may explain the dearth of cancer-specific mutations in these proteins, because these non-alpha isoforms could contribute to the oncogenic phenotype of the cell by differential expression.
Asunto(s)
Transformación Celular Neoplásica , Fosfatidilinositol 3-Quinasas/química , Animales , Antibióticos Antineoplásicos/farmacología , Western Blotting , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Embrión de Pollo , Fosfatidilinositol 3-Quinasa Clase I , Medio de Cultivo Libre de Suero/farmacología , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Plásmidos/metabolismo , Mutación Puntual , Unión Proteica , Isoformas de Proteínas , Proteínas Quinasas/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Factores de Tiempo , Transfección , Proteínas ras/metabolismoRESUMEN
The catalytic subunit p110alpha of the phosphoinositide 3-kinase (PI3K) and the serine-threonine protein kinase Akt have been extensively studied as retroviral oncoproteins. The experimental tools developed with the retroviral vectors are now being applied to PI3K mutations in human cancer. The most frequently occurring mutants of p110alpha are oncogenic in vitro and in vivo, show gain of enzymatic function, activate Akt, and their oncogenic activity is sensitive to rapamycin. The related isoforms p110beta, gamma and delta induce oncogenic transformation as wild-type proteins. Mutated p110alpha proteins are ideal drug targets. Identification of small molecule inhibitors that specifically target these mutant proteins is a realistic and urgent goal.
Asunto(s)
Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sirolimus/farmacología , Animales , Virus del Sarcoma Aviar/enzimología , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/fisiología , Mutación , Proteínas Nucleares , Proteína Oncogénica v-akt/metabolismo , Proteínas Oncogénicas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/metabolismo , Serina-Treonina Quinasas TOR , Proteína 1 de Unión a la Caja YRESUMEN
There have long been indications of a role for PI3K (phosphatidylinositol 3-kinase) in cancer pathogenesis. Experimental data document a requirement for deregulation of both transcription and translation in PI3K-mediated oncogenic transformation. The recent discoveries of cancer-specific mutations in PIK3CA, the gene that encodes the catalytic subunit p110alpha of PI3K, have heightened the interest in the oncogenic potential of this lipid kinase and have made p110alpha an ideal drug target.
Asunto(s)
Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/genética , Dominio Catalítico , Factores de Transcripción Forkhead/genética , Humanos , Mutación , Neoplasias/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/genética , Transcripción GenéticaRESUMEN
The PI3K signaling pathway is upregulated in numerous cancers. The catalytic subunit p110alpha of PI3K shows hot spot mutations in nearly 30% of several types of solid tumors. The most prominent of these mutations result in gain of enzymatic function, activate Akt signaling and induce oncogenic cellular transformation. The mutated p110alpha proteins are ideal targets for specific small molecule inhibitors that discriminate between the oncogenic and the wild-type forms of the enzyme. Such inhibitors could become highly effective anti-cancer drugs.
Asunto(s)
Mutación , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Animales , Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Transducción de SeñalRESUMEN
Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.
Asunto(s)
Transformación Celular Neoplásica/genética , Mutación Missense , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Animales , Proteínas Portadoras/metabolismo , Proliferación Celular , Embrión de Pollo , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Neoplasias/enzimología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/genéticaRESUMEN
L35 and FAO cells were derived as single cell isolates from H35 cells. Whereas L35 cells do not express microsomal triglyceride transfer protein (MTP), which regulates lipoprotein secretion, they express CYP7A1, which regulates bile acid synthesis from cholesterol. FAO cells display the opposite phenotype (i.e. expression of MTP but not CYP7A1). We examined the molecular basis of the transcriptional inactivation of the MTP gene in L35 cells. Nested deletion and mutagenesis studies show that a conserved DR1 element within the 135-bp proximal MTP promoter is responsible for differential expression by L35 and FAO cells. Yeast one-hybrid screening identified apolipoprotein A1 regulatory protein-1/chicken ovalbumin upstream promoter transcription factor II (ARP-1/COUP-TFII) and retinoid X receptor (RXRalpha) as the protein factors that can bind to the conserved DR1 element. Nuclear extracts from L35 cells contained 2-fold more ARP-1/COUP-TFII and 50% less RXRalpha than those from FAO cells. Immunologic studies show that in L35 cells, ARP-1/COUP-TFII is bound to the DR1 element, whereas in FAO cells, a complex containing RXRalpha is bound to the DR1 element. Co-transfection studies show that ARP-1/COUP-TFII repressed MTP promoter activity by approximately 70% in FAO hepatoma cells, whereas RXRalpha and its ligand 9-cis-retinoic acid increased MTP promoter activity by 6-fold in L35 cells. The combined data suggest that in the context of the MTP promoter, ARP-1/COUP-TFII (repressor) and a complex containing RXRalpha (inducer) compete for the DR1 element. Analysis of the CYP7A1 promoter revealed that it is approximately 5-fold more active in L35 cells than in FAO cells. Co-transfection of an ARP-1/COUP-TFII expression vector showed that it enhances CYP7A1 promoter activity by 6-fold in FAO cells. These combined findings indicate that ARP-1/COUP-TFII acts as both a transcriptional repressor (of MTP) and as a transcription activator (of CYP7A1). This dual function of ARP-1/COUP-TFII may play an important role in determining the metabolic phenotype of individual liver cells.
Asunto(s)
Carcinoma Hepatocelular , Proteínas Portadoras/genética , Colesterol 7-alfa-Hidroxilasa/genética , Neoplasias Hepáticas , Activación Transcripcional/fisiología , Animales , Secuencia de Bases , Regulación Neoplásica de la Expresión Génica , Prueba de Complementación Genética , Ratones , Datos de Secuencia Molecular , Fenotipo , Regiones Promotoras Genéticas/genética , Ratas , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Despite a complete lack of microsomal triglyceride transfer protein (MTP), L35 rat hepatoma cells secrete triglyceride-containing lipoproteins, albeit at a rate 25% of that of parental FAO hepatoma cells, which express high levels of MTP. The inability to express MTP was associated with a complete block in the secretion of both apolipoprotein (apo)B-100 and apoB-48. Stable expression of a MTP transgene restored the secretion of both apoB-100 and apoB-48 in L35 cells, indicating that MTP is essential for the secretion of both forms of apoB. Treatment with the MTP inhibitor BMS-200150 reduced the secretion of triglyceride by 70% in FAO cells, whereas the inhibitor did not affect the secretion of triglycerides by L35 cells. Thus, in the presence of the MTP inhibitor, both cell types secreted triglycerides at similar rates. Essentially, all of the triglycerides secreted by L35 cells were associated with HDL containing apoA-IV and apoE but devoid of apoB-100 or apoB-48. These results suggest that these triglyceride-containing lipoproteins are assembled and secreted via a pathway that is independent of both apoB and MTP. Our findings support the concept that apoB and MTP co-evolved and provided a means to augment the secretion of triglyceride through the formation of lipoproteins containing large hydrophobic cores enriched with triglycerides.