RESUMEN

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL). The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment. The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death. Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.

Asunto(s)

Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/biosíntesis , Inhibidores Enzimáticos/farmacología , Leucemia de Células T/metabolismo , Proteínas Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Humanos , Células Jurkat , Leucemia de Células T/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo

RESUMEN

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.

Asunto(s)

Neoplasias de la Mama/enzimología , Colina Quinasa/biosíntesis , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Colina Quinasa/antagonistas & inhibidores , Estudios de Cohortes , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Espectroscopía Infrarroja Corta , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

AIMS: Malignant transformation results in overexpression of choline-kinase (CHK) and altered choline metabolism, which is potentially detectable by immunohistochemistry (IHC). We investigated the utility of CHK-alpha (CHKA) IHC as a complement to current diagnostic investigation of prostate cancer by analysing expression patterns in normal (no evidence of malignancy) and malignant human prostate tissue samples. METHODS: As an initial validation, paraffin-embedded prostatectomy specimen blocks with both normal and malignant prostate tissue were analysed for CHKA protein and mRNA expression by western blot and quantitative reverse transcriptase PCR (qRT-PCR), respectively. Subsequently, 100 paraffin-embedded malignant prostate tumour and 25 normal prostate cores were stained for both Ki67 (labelling-index: LI) and CHKA expression. RESULTS: The validity of CHKA-antibody was verified using CHKA-transfected cells and siRNA knockdown. Immunoblotting of tissues showed good resolution of CHKA protein in malignant prostate, verifying use of the antibody for IHC. There was minimal qRT-PCR detectable CHKA mRNA in normal tissue, and conversely high expression in malignant prostate tissues. IHC of normal prostate cores showed mild (intensity) CHKA expression in only 28% (7/25) of samples with no Ki67 expression. In contrast, CHKA was expressed in all malignant prostate cores along with characteristically low proliferation (median 2% Ki67-LI; range 1-17%). Stratification of survival according to CHK intensity showed a trend towards lower progression-free survival with CHK score of 3. CONCLUSIONS: Increased expression of CHKA, detectable by IHC, is seen in malignant lesions. This relatively simple cost-effective technique (IHC) could complement current diagnostic procedures for prostate cancer and, therefore, warrants further investigation.

Asunto(s)

Biomarcadores de Tumor/análisis , Colina Quinasa/biosíntesis , Neoplasias de la Próstata/enzimología , Western Blotting , Colina Quinasa/análisis , Supervivencia sin Enfermedad , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Pronóstico , Neoplasias de la Próstata/mortalidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Lipid composition in cell membrane is closely associated with cell characteristics. Here, matrix-assisted laser desorption/ionization- Fourier transform ion cyclotron resonance mass spectrometry was employed to in situ determine membrane components of human mammary epithelial cells (MCF-10 A) and six different breast cancer cell lines (i.e., BT-20, MCF-7, SK-BR-3, MDA-MB-231, MDA-MB-157, and MDA-MB-361) without any lipid extraction and separation. Partial least-square discriminant analysis indicated that changes in the levels of these membrane lipids were closely correlated with the types of breast cell lines. Elevated levels of polyunsaturated lipids in MCF-10 A cells relative to six breast cancer cells and in BT-20 cells relative to other breast cancer cell lines were detected. The Western blotting assays indicated that the expression of five lipogenesis-related enzymes (i.e., fatty acid synthase 1(FASN1), stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 5 (SCD5), choline kinase α (CKα), and sphingomyelin synthase 1) was associated with the types of the breast cells, and that the SCD1 level in MCF-7 cells was significantly increased relative to other breast cell lines. Our findings suggest that elevated expression levels of FASN1, SCD1, SCD5, and CKα may closely correlated with enhanced levels of saturated and monounsaturated lipids in breast cancer cell lines.

Asunto(s)

Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Línea Celular Tumoral , Colina Quinasa/biosíntesis , Colina Quinasa/genética , Acido Graso Sintasa Tipo I/biosíntesis , Acido Graso Sintasa Tipo I/genética , Femenino , Humanos , Lipogénesis/genética , Células MCF-7 , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estearoil-CoA Desaturasa/biosíntesis , Estearoil-CoA Desaturasa/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética

RESUMEN

Cancer cells have distinct metabolomic profile. Metabolic enzymes regulate key oncogenic signaling pathways and have an essential role on tumor progression. Here, serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers. The results showed that dysregulation of choline metabolism occurred in TCL and was related to tumor cell overexpression of choline kinase-α (Chokα). In T-lymphoma cells, pharmacological and molecular silencing of Chokα significantly decreased Ras-GTP activity, AKT and ERK phosphorylation and MYC oncoprotein expression, leading to restoration of choline metabolites and induction of tumor cell apoptosis/necropotosis. In a T-lymphoma xenograft murine model, Chokα inhibitor CK37 remarkably retarded tumor growth, suppressed Ras-AKT/ERK signaling, increased lysophosphatidylcholine levels and induced in situ cell apoptosis/necropotosis. Collectively, as a regulatory gene of aberrant choline metabolism, Chokα possessed oncogenic activity and could be a potential therapeutic target in TCL, as well as other hematological malignancies with interrupted Ras signaling pathways.

Asunto(s)

Colina Quinasa/biosíntesis , Colina/sangre , Linfoma de Células T/sangre , Adulto , Anciano , Animales , Apoptosis/genética , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/sangre , Inhibidores Enzimáticos/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células T/genética , Linfoma de Células T/patología , Masculino , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Metabolic rearrangements subsequent to malignant transformation are not well characterized in endometrial cancer. Identification of altered metabolites could facilitate imaging-guided diagnosis, treatment surveillance, and help to identify new therapeutic options. Here, we used high-resolution magic angle spinning magnetic resonance mass spectroscopy on endometrial cancer surgical specimens and normal endometrial tissue to investigate the key modulators that might explain metabolic changes, incorporating additional investigations using qRT-PCR, Western blotting, tissue microarrays (TMA), and uptake assays of [(3)H]-labeled choline. Lipid metabolism was severely dysregulated in endometrial cancer with various amino acids, inositols, nucleobases, and glutathione also altered. Among the most important lipid-related alterations were increased phosphocholine levels (increased 70% in endometrial cancer). Mechanistic investigations revealed that changes were not due to altered choline transporter expression, but rather due to increased expression of choline kinase α (CHKA) and an activated deacylation pathway, as indicated by upregulated expression of the catabolic enzymes LYPLA1, LYPLA2, and GPCPD1. We confirmed the significance of CHKA overexpression on a TMA, including a large series of endometrial hyperplasia, atypical hyperplasia, and adenocarcinoma tissues, supporting a role for CHKA in malignant transformation. Finally, we documented several-fold increases in the uptake of [(3)H]choline in endometrial cancer cell lines compared with normal endometrial stromal cells. Our results validate deregulated choline biochemistry as an important source of noninvasive imaging biomarkers for endometrial cancer.

Asunto(s)

Colina Quinasa/biosíntesis , Colina/metabolismo , Neoplasias Endometriales/metabolismo , Fosfolípidos/metabolismo , Adenocarcinoma/metabolismo , Línea Celular , Línea Celular Tumoral , Colina Quinasa/metabolismo , Neoplasias Endometriales/enzimología , Femenino , Humanos , Hiperplasia/metabolismo , Metabolismo de los Lípidos , Persona de Mediana Edad , Fosfolipasas/metabolismo , Fosforilcolina/metabolismo , Transducción de Señal , Tioléster Hidrolasas/metabolismo

RESUMEN

INTRODUCTION: Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. METHODS: Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson's correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. RESULTS: Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. CONCLUSION: The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.

Asunto(s)

Neoplasias de la Mama/metabolismo , Colina/metabolismo , Glicerilfosforilcolina/metabolismo , Fosforilcolina/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Colina Quinasa/biosíntesis , Colina Quinasa/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metabolómica , Ratones , Trasplante de Neoplasias , Hidrolasas Diéster Fosfóricas/biosíntesis , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Análisis de Matrices Tisulares , Transcriptoma , Trasplante Heterólogo

RESUMEN

Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-ß (C/EBPß) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPß expression. Elevated levels of C/EBPß bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/genética , Diferenciación Celular/efectos de los fármacos , Neuronas/metabolismo , Fosfolípidos/biosíntesis , Animales , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Proliferación Celular , Colina Quinasa/biosíntesis , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Humanos , Ratones , Neuritas/metabolismo , Neuronas/citología , Fosfatidilcolinas/metabolismo , Fosfolípidos/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Tretinoina/farmacología

RESUMEN

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length (∼70-kDa) TgCK displayed a low affinity for choline (K(m) ∼0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by ∼60%, which was abolished in the off state of the mutant (Δtgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.

Asunto(s)

Colina Quinasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Mutagénesis , Fosfatidilcolinas/biosíntesis , Proteínas Protozoarias/biosíntesis , Toxoplasma/enzimología , Secuencia de Bases , Colina Quinasa/genética , Deanol/metabolismo , Datos de Secuencia Molecular , Mutación , Fosfatidilcolinas/genética , Multimerización de Proteína/fisiología , Proteínas Protozoarias/genética , Toxoplasma/genética

RESUMEN

Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. Increased phosphorylation of choline is a hallmark characteristic of the malignant phenotype in a variety of neoplasms. However, in hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase α (ChKα), in hypoxic PC-3 human prostate cancer cells. Hypoxia decreased choline phosphorylation, choline kinase activity, and ChKα mRNA and protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved DNA consensus binding motif, hypoxia response element-7 (HRE7), at position -222 relative to +1 translation start site, for binding the hypoxia dependent master regulator transcription factor, hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic DNA and cloned into a luciferase-based reporter vector system. In PC-3 cells, hypoxia decreased the expression of luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this hypoxia effect, further demonstrating the involvement of HRE7 in hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.

Asunto(s)

Hipoxia de la Célula/fisiología , Colina Quinasa/metabolismo , Colina/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Colina Quinasa/biosíntesis , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/metabolismo , Elementos de Respuesta/genética , Transcripción Genética

RESUMEN

PURPOSE: To evaluate the accuracy and biological basis for [(11)C]choline-PET-CT in the nodal staging of high risk localized prostate cancer patients. EXPERIMENTAL DESIGN: Twenty-eight patients underwent dynamic [(11)C]choline-PET-CT of the pelvis and lower abdomen prior to extended laparoscopic pelvic lymph node dissection (eLPL). The sensitivity and specificity of [(11)C]choline PET, [(11)C]choline PET-CT, and MRI for nodal detection were calculated. Average and maximal standardized uptake values (SUV(ave), SUV(max)) were compared with choline kinase alpha (CHKα) and Ki67 immunohistochemistry scores. RESULTS: Four hundred and six lymph nodes (LN), in 26 patients, were assessable. Twenty-seven (6.7%) involved pelvic nodes at eLPL were detected in 9 patients. Seventeen of the 27 involved nodes were subcentimeter. The sensitivity and specificity on a per nodal basis were 18.5% and 98.7%, 40.7% and 98.4%, and 51.9% and 98.4% for MRI, [(11)C]choline PET, and [(11)C]choline PET-CT, respectively. Sensitivity was higher for [(11)C]choline PET-CT compared with MRI (P = 0.007). A higher nodal detection rate, including subcentimeter nodes, was seen with [(11)C]choline PET-CT than MRI. Malignant lesions showed CHKα expression in both cytoplasm and nucleus. SUV(ave) and SUV(max) strongly correlated with CHKα staining intensity (r = 0.68, P < 0.0001 and r = 0.63, P = 0.0004, respectively). In contrast, Ki67 expression was generally low in all tumors. CONCLUSION: This study establishes the relationship between [(11)C]choline PET-CT uptake with choline kinase expression in prostate cancer and allows it to be used as a noninvasive means of staging pelvic LNs, being highly specific and more sensitive than MRI, including the detection of subcentimeter disease.

Asunto(s)

Colina Quinasa/biosíntesis , Metástasis Linfática/diagnóstico , Imagen Multimodal/métodos , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Anciano , Anciano de 80 o más Años , Radioisótopos de Carbono , Colina/farmacocinética , Humanos , Inmunohistoquímica , Límite de Detección , Ganglios Linfáticos/diagnóstico por imagen , Ganglios Linfáticos/patología , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pelvis , Estudios Prospectivos , Próstata/diagnóstico por imagen , Próstata/enzimología , Neoplasias de la Próstata/patología , Reproducibilidad de los Resultados

RESUMEN

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

Asunto(s)

Colina Quinasa/metabolismo , Colina/metabolismo , Furanos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilcolina/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Línea Celular Tumoral , Colina Quinasa/biosíntesis , Colina Quinasa/genética , Regulación hacia Abajo/efectos de los fármacos , Células HCT116 , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Proteínas de la Membrana/deficiencia , Fosfohidrolasa PTEN/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo

RESUMEN

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKalpha (but not ChoKbeta) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC biosynthesis and degradation, respectively.

Asunto(s)

Neoplasias Ováricas/enzimología , Fosfatidilcolinas/biosíntesis , Colina Quinasa/biosíntesis , Colina Quinasa/genética , Colina Quinasa/metabolismo , Activación Enzimática , Células Epiteliales/patología , Femenino , Humanos , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosfolipasa D/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Elevated phosphocholine (PC) and total choline (tCho) metabolites are widely established characteristics of most cancer cells, including breast cancer. Effective silencing of choline kinase (chk), the enzyme that converts choline to PC, is associated with reduced tumor growth. The functional importance and down-regulation of chk using RNA interference has been previously established. Here, we report on the preclinical evaluation of lentiviral vector-mediated down-regulation of chk using short hairpin RNA (shRNA) in established tumors derived from human breast cancer cells. Concentrated lentivirus expressing shRNA against chk was injected i.v. in the tail vein of MDA-MB-231 tumor-bearing female severe combined immunodeficient mice. Transduction efficiency in cells and tumors in vivo was assessed optically by enhanced green fluorescent protein expression and additionally from chk mRNA and protein levels. An 80% reduction in chk mRNA and protein was achieved following approximately 90% transduction efficiency in cells. After transduction with chk-shRNA, (1)H magnetic resonance spectroscopy (MRS) of cell and tumor extracts showed decreases in PC and tCho levels (P < 0.01 and 0.05, respectively) in comparison with controls. PC levels were monitored noninvasively by (31)P MRS in tumors and by (1)H MRS in cell and tumor tissue extracts. Noninvasive (31)P MR spectra of chk-shRNA-transduced tumors in vivo showed lower PC and phosphomonoester levels that were associated with reduced tumor growth and proliferation. This study shows the use of lentiviral vectors to target chk in a human breast cancer xenograft and noninvasive MRS detection of this targeting.

Asunto(s)

Neoplasias de la Mama/terapia , Colina Quinasa/deficiencia , Colina Quinasa/genética , Terapia Genética/métodos , ARN Interferente Pequeño/genética , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Colina Quinasa/biosíntesis , Colina Quinasa/metabolismo , Regulación hacia Abajo , Femenino , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Ratones , Ratones SCID , Fosforilcolina/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción Genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

In the yeast Saccharomyces cerevisiae, the CKI1-encoded choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine via the CDP-choline branch of the Kennedy pathway. Analysis of a P(CKI1)-lacZ reporter gene revealed that CKI1 expression was regulated by intracellular levels of the essential mineral zinc. Zinc depletion resulted in a concentration-dependent induction of CKI1 expression. This regulation was mediated by the zinc-sensing and zinc-inducible transcriptional activator Zap1p. A purified Zap1p probe interacted with two putative UAS(ZRE) sequences (ZRE1 and ZRE2) in the CKI1 promoter. Mutations of ZRE1 and ZRE2 to a nonconsensus UAS(ZRE) attenuated the induction of CKI1 expression in response to zinc depletion. A UAS(INO) element in the CKI1 promoter was responsible for stimulating CKI1 expression, but this element was not involved with the regulation by zinc depletion. The induction of CKI1 expression in zinc-depleted cells translated into increased choline kinase activity in vitro and in vivo, and an increase in phosphatidylcholine synthesis via the Kennedy pathway.

Asunto(s)

Colina Quinasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transactivadores/metabolismo , Zinc/farmacología , Colina Quinasa/genética , Relación Dosis-Respuesta a Droga , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/genética , Elementos de Respuesta/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genética , Factores de Transcripción , Zinc/deficiencia

RESUMEN

Carcinogenesis is a long process that results in the accumulation of genetic alterations primarily in genes involved in the regulation of signalling pathways relevant for the regulation of cell growth and the cell cycle. Alteration of additional genes regulating cell adhesion and migration, angiogenesis, apoptosis, and drug resistance confers to the cancer cells a more malignant phenotype. Genes that participate in the regulation of some critical metabolic pathways are also altered during this process. Choline kinase (ChoK) has been reported to belong to the latter family of cancer-related genes. Recently, we have reported that increased activity of ChoK is observed in human breast carcinomas. Here, we provide further evidence that ChoK dysregulation is a frequent event found in a variety of human tumors such as lung, colorectal, and prostate tumors. Furthermore, a large panel of human tumor-derived cell lines also show increased ChoK activity when compared to appropriate non-tumorigenic or primary cells. These findings strongly support the role of ChoK alterations in the carcinogenic process in human tumors, suggesting that ChoK could be used as a tumor marker.

Asunto(s)

Colina Quinasa/biosíntesis , Neoplasias Colorrectales/enzimología , Neoplasias Pulmonares/enzimología , Neoplasias de la Próstata/enzimología , Adulto , Anciano , Biomarcadores de Tumor/análisis , Carcinoma/enzimología , Células Cultivadas , Colon/enzimología , Humanos , Pulmón/enzimología , Masculino , Persona de Mediana Edad , Próstata/enzimología , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

Ras proteins are molecular switches that control signaling pathways critical in the onset of a variety of human cancers. The signaling pathways activated by Ras proteins are those controlled by its direct effectors such as the serine-threonine protein kinase Raf-1, the exchange factor for other GTPases Ral-GDS, and the lipid kinase PI3K. As a consequence of Ras activation, a number of additional enzymes are affected, including several members of the serine-threonine intracellular proteins kinases as well as enzymes related to phospholipid metabolism regulation such as phospholipases A2 and D, and choline kinase. The precise mechanisms by which ras oncogenes impinge into these later molecules and their relevance to the onset of the carcinogenic process is still not fully understood. Here we have investigated the mechanism of regulation of choline kinase by Ras proteins and found no direct link between PLD and choline kinase activation. We provide evidence that Ras proteins regulate the activity of choline kinase through its direct effectors Ral-GDS and PI3K, while the Raf pathways seems to be not relevant in this process. The importance of Ras-dependent activation of choline kinase is discussed.

Asunto(s)

Colina Quinasa/biosíntesis , Factor de Intercambio de Guanina Nucleótido ral/fisiología , Proteínas ras/fisiología , Células 3T3/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Colina Quinasa/genética , Embrión de Mamíferos , Activación Enzimática , Inducción Enzimática , Genes ras , Guanosina Trifosfato/fisiología , Humanos , Isoenzimas/fisiología , Riñón/citología , Ratones , Mutación Missense , Fosfolipasa D/genética , Fosfolipasa D/fisiología , Fosforilcolina/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/fisiología , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteínas Recombinantes de Fusión/fisiología , Sistemas de Mensajero Secundario , Transfección

RESUMEN

The Saccharomyces cerevisiae CPT1 and EPT1 genes encode distinct choline- and choline/ethanolaminephosphotransferases, respectively. In vitro, each gene product accounts for 50% of the measurable choline-phosphotransferase activity. Strains containing null mutations in the CPT1 and EPT1 loci were used to investigate the function of each gene product in vivo. The CPT1 gene product was responsible for 95% of phosphatidylcholine (PC) synthesis via the CDP-choline pathway in vivo. The EPT1 gene product accounted for only 5% of PC synthesis in vivo. Chimeric CPT1/EPT1 enzymes with diacylglycerol and CDP-aminoalcohol specificities both similar and distinct from the parental enzymes were used to determine the specific segments of the CPT1/EPT1 gene products required to restore PC synthesis to cpt- cells in vivo. Only chimeras expressing the CDP-aminoalcohol specificity region of CPT1 were capable of PC synthesis via the CDP-choline pathway in vivo. Analysis of phospholipids extracted from wild type, cpt-, and ept- cells labeled with 32Pi indicated an intact CPT1 gene product was required for the pleiotropic regulation of phospholipid synthesis by inositol. Chimeric CPT1/EPT1 enzymes expressed in a cpt- background mapped the regulatory region of the CPT1 gene product required for the inositol-dependent regulation of phospholipid synthesis to the CDP-aminoalcohol binding domain of CPT1. Strains harboring dysfunctional cholinephosphotransferase enzymes also displayed decreased levels of choline uptake, suggesting that a feedback loop exists to coordinate choline uptake with ongoing PC biosynthesis. The data also implicate the CPT1 gene product in PC biosynthesis from an endogenous source of choline derived from turnover of PC via the phosphatidylserine-dependent route for PC synthesis.

Asunto(s)

Colina Quinasa/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Genes Fúngicos , Fosfatidilcolinas/biosíntesis , Saccharomyces cerevisiae/metabolismo , Colina/metabolismo , Colina Quinasa/biosíntesis , Diacilglicerol Colinafosfotransferasa/biosíntesis , Cinética , Fosfatos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Serina/metabolismo , Fracciones Subcelulares/enzimología , Especificidad por Sustrato

RESUMEN

The present study documents the patterns of mRNA expression for the ceramide galactosyltransferase (CGT), the CTP:phosphocholine cytidylyltransferase (CT), and the choline kinase (CK) during the myelination period of the mouse central nervous system (CNS) and peripheral nervous system (PNS). Using the Northern blot technique with densitometric analyses, we show that the CK gene is not developmentally regulated during the period studied, whereas a peak of expression of the CT gene is observed around day 10. On the other hand, the expression of the CGT gene is similar to that of the MBP gene in the CNS and the PNS. Therefore, the synthesis of the galactosylceramides during the myelination period seems to be controlled at the level of the expression of the CGT gene. These results were compared to those of a neurological mutant, the trembler mouse, whose PNS myelination is deficient. Our results clearly indicate that the deficit in the accumulation of the galactosylceramides documented for this mutant is well correlated to a reduced CGT gene expression.

Asunto(s)

Encéfalo/metabolismo , Colina Quinasa/biosíntesis , Galactosiltransferasas/biosíntesis , Expresión Génica , Proteína Básica de Mielina/biosíntesis , Vaina de Mielina/fisiología , Nucleotidiltransferasas/biosíntesis , Nervio Ciático/metabolismo , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Citidililtransferasa de Colina-Fosfato , Cartilla de ADN , Sondas de ADN , Ratones , Ratones Endogámicos CBA , Ratones Mutantes Neurológicos , Datos de Secuencia Molecular , N-Acilesfingosina Galactosiltransferasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Valores de Referencia , Nervio Ciático/crecimiento & desarrollo

RESUMEN

The yeast phosphatidylethanolamine methylation pathway is encoded by two structural genes, PEM1 and PEM2. The abundance of their transcripts was coordinately repressed by myo-inositol and choline. The most upstream transcriptional start sites for PEM1 and PEM2 were mapped at positions -142 and -42 relative to their first ATG codons, respectively. Promoter deletion analysis defined the 5' boundary of the regulatory region of PEM1 between -336 and -332 and that of PEM2 between -177 and -158. The 38-bp sequence between -336 and -299 from PEM1 and the 48-bp sequence between -177 and -130 from PEM2 conferred regulated transcription upon an upstream-activation-sequence-deficient test gene, CYC1-lacZ. Comparison of these two regions revealed the presence of a common octameric sequence, 5-CATRTGAA-3', which occurred twice in the 38-bp PEM1 regulatory region and once, followed by the 5'-AAACCCACACATG-3' GRFI site, in the 48-bp PEM2 regulatory region. When synthesized chemically and placed in front of CYC1-lacZ, a single copy of CATATGAA directed a rather low level of gene expression, but multiple copies produced high-level expression. In both cases, gene expression was sensitive to myo-inositol and choline. The synthesized GRFI site directed considerable but constitute lacZ expression. When used in conjunction with CATATGAA, synergistic, regulated gene expression was obtained. Hence CATRTGAA was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2. Binding proteins to these sequences were demonstrated by electrophoretic mobility shift assay. Protein binding to CATRTGAA was not competitive with binding to the GRFI sequence, and vice versa. CATRTGAA was also found in the upstream regions of other genes encoding phospholipid-synthesizing enzymes, such as choline kinase, phosphatidylserine synthase, and myo-inositol-1-phosphate synthase, known to be repressed by myo-inositol and choline.

Asunto(s)

CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Colina Quinasa/genética , Regulación Enzimológica de la Expresión Génica , Metiltransferasas/metabolismo , Mio-Inositol-1-Fosfato Sintasa/genética , Fosfatidiletanolaminas/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Bases , Sitios de Unión , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/biosíntesis , Colina/farmacología , Colina Quinasa/biosíntesis , Secuencia de Consenso , Inositol/farmacología , Metilación , Datos de Secuencia Molecular , Mio-Inositol-1-Fosfato Sintasa/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos