RESUMEN
The molecular mechanisms underlying spontaneous neoplastic transformation in cultured mammalian cells remain poorly understood, confounding recognition of parallels with the biology of naturally occurring cancer. The broad use of tumorigenic canine cell lines as research tools, coupled with the accumulation of cytogenomic data from naturally occurring canine cancers, makes the domestic dog an ideal system in which to investigate these relationships. We developed a canine kidney cell line, CKB1-3T7, which allows prospective examination of the onset of spontaneous immortalization and tumorigenicity. We documented the accumulation of cytogenomic aberrations in CKB1-3T7 over 24 months in continuous culture. The majority of aberrations emerged in parallel with key phenotypic changes in cell morphology, growth kinetics, and tumor incidence and latency. Focal deletion of CDKN2A/B emerged first, preceding the onset and progression of tumorigenic potential, and progressed to a homozygous deletion across the cell population during extended culture. Interestingly, CKB1-3T7 demonstrated a tumorigenic phenotype in vivo prior to exhibiting loss of contact inhibition in vitro. We also performed the first genome-wide characterization of the canine tumorigenic cell line MDCK, which also exhibited CDKN2A/B deletion. MDCK and CKB1-3T7 cells shared several additional aberrations that we have reported previously as being highly recurrent in spontaneous canine cancers, many of which, as with CDKN2A/B deletion, are evolutionarily conserved in their human counterparts. The conservation of these molecular events across multiple species, in vitro and in vivo, despite their contrasting karyotypic architecture, is a powerful indicator of a common mechanism underlying emerging neoplastic activity. Through integrated cytogenomic and phenotypic characterization of serial passages of CKB1-3T7 from initiation to development of a tumorigenic phenotype, we present a robust and readily accessible model (to be made available through the American Type Culture Collection) of spontaneous neoplastic transformation that overcomes many of the limitations of earlier studies.
Asunto(s)
Transformación Celular Neoplásica/genética , Aberraciones Cromosómicas , Cariotipo , Neoplasias/genética , Animales , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Cultivadas , Variaciones en el Número de Copia de ADN , Perros , Hibridación Fluorescente in Situ , Células de Riñón Canino Madin Darby , Masculino , Neoplasias/patologíaRESUMEN
The pharmacological properties of fatty acid amidohydrolase (FAAH) at different assay pH values were investigated using [(3)H]-anandamide ([(3)H]-AEA) as substrate in rat brain homogenates and in COS-1 [corrected] cells transfected with wild type and mutant FAAH. Rat brain hydrolysis of [(3)H]-AEA showed pH dependency with an optimum around pH 8-9. Between pH 6.3 and 8.2, the difference in activity was due to differences in the V(max), rather than the K(M) values. For inhibition of rat brain [(3)H]-AEA metabolism by a series of known FAAH inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethylsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoylserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of FAAH. The K(i)((slope)) and K(i)((intercept)) values for (-)ibuprofen at pH 5.28 were 11 and 143 microM, respectively. At pH 8.37, the corresponding values were 185 and 3950 microM, respectively. The pH dependency for the inhibition by OTMK and (-)ibuprofen was also seen in COS-1 [corrected] cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant enzymes were seen. It is concluded that the pharmacological properties of FAAH are highly pH-dependent. The higher potency of ibuprofen at lower pH values raises the possibility that in certain types of inflamed tissue, the concentration of this compound following oral administration may be sufficient to inhibit FAAH.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Encéfalo/efectos de los fármacos , Ibuprofeno/farmacología , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Encéfalo/enzimología , Células COS , Endocannabinoides , Concentración de Iones de Hidrógeno , Hidrólisis , Técnicas In Vitro , Masculino , Mutación , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Fatty acid amide hydrolase is an integral membrane protein that hydrolyzes a novel and growing class of neuromodulatory fatty acid molecules, including anandamide, 2-arachidonyl glycerol, and oleamide. This activity is inhibited by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine residues were mutated to alanine and the effects on activity were determined. Mutants were prepared using site-directed mutagenesis methods and expressed in COS-7 cells. Serine mutations S217A and S241A completely abolished enzymatic activity. Mutants S152A and C249A had no effect on activity, while S218A showed a slight decrease in activity. To confirm these results biochemically, the mutant enzymes were reacted with the irreversible inhibitor [(14)C]-diisopropyl fluorophosphate. All of the mutants except S217A and S241A were labeled. We therefore confirm that fatty acid amide hydrolase is a serine hydrolase and propose that both Ser-217 and Ser-241 are essential for enzyme activity.
Asunto(s)
Amidohidrolasas/genética , Serina/química , Alanina/química , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Catálisis , Cisteína/química , Isoflurofato , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfolipasas A/metabolismo , Inhibidores de Proteasas , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Anandamide amidase is the hydrolytic enzyme responsible for the breakdown of anandamide, an endogenous cannabimimetic, to arachidonate and ethanolamine. Another enzymatic activity called anandamide synthase catalyzes the reverse reaction, that is the condensation of arachidonate and ethanolamine. Using a recently cloned rat fatty acid amidohydrolase (FAAH), we tested the hypothesis that the synthase and the amidase activities are catalyzed by the same enzyme. Untransfected and vector transfected (pcDNA3) COS-7 cells did not express detectable levels of either the amidase or synthase. However, when COS-7 cells were transiently transfected with a rat FAAH pcDNA3 construct, both amidase and synthase were concomitantly expressed. These results indicate that the enzymatic formation of anandamide from arachidonic acid and ethanolamine can be mediated by anandamide amidase acting in the reverse direction. The FAAH transfected cells expressed higher levels of enzyme than either rat brain homogenates or neuroblastoma cells in culture. Furthermore, the reaction rate for the amidase in FAAH transfected COS-7 cells, neuroblastoma cells and brain homogenate was always greater than the synthase reaction. These studies raise the question if this synthase reaction serves any physiological role, especially in view of the evidence that anandamide can be formed by a different pathway.
Asunto(s)
Amidohidrolasas/metabolismo , Ácido Araquidónico/metabolismo , Etanolamina/metabolismo , Animales , Encéfalo/metabolismo , Células COS , Catálisis , Clonación Molecular , Hidrólisis , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonoyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described (Koutek et al., J Biol Chem 269: 22937-22940, 1994). In the current study, methyl arachidonyl fluorophosphonate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approximately 1000-fold more potent than Arach-CF3. MAFP demonstrated selectivity towards anandamide amidase for which it was approximately 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, respectively. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addition, can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonoyl ethanolamide amidase.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Inhibidores Enzimáticos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sitios de Unión , Ciclohexanoles/metabolismo , Inhibidores Enzimáticos/metabolismo , Organofosfonatos , Ratas , Receptores de Cannabinoides , Receptores de Droga/metabolismoRESUMEN
Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (palmitylsulfonyl fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50 values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylfluoride had an IC50 of 18 nM and C20 sulfonylfluoride had an IC50 of 78 microM. The C14, C16, and C18 sulfonyl fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Ácidos Araquidónicos/metabolismo , Cannabinoides/metabolismo , Ácidos Grasos/farmacología , Palmitatos/farmacología , Receptor Cannabinoide CB2 , Receptores de Droga/metabolismo , Sulfonas/farmacología , Animales , Encéfalo/metabolismo , Endocannabinoides , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/química , Neuronas/metabolismo , Palmitatos/química , Fluoruro de Fenilmetilsulfonilo/análogos & derivados , Fluoruro de Fenilmetilsulfonilo/farmacología , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides , Relación Estructura-Actividad , Sulfonas/química , Células Tumorales CultivadasRESUMEN
Arachidonoyl ethanolamide-[1,2-14C] was prepared and evaluated as a substrate for anandamide amidase in a radioenzymatic assay that does not require a thin layer chromatography separation step. Using this substrate the release of ethanolamine-[1,2-14C] is linear for approximately thirty minutes. Anandamide amidase exhibits maximal activity between pH 8 and pH 9 with a steep decline in activity at pH values below 6 and above 10. Arachidonoyl ethanolamide-[1,2-14C] was used for the assay of anandamide amidase from 10 micrograms to 100 micrograms protein, from cow brain homogenate, in a 0.2 ml incubation mixture. When plotted as a rectangular hyperbola of the steady-state Michaelis-Menten equation, an approximate Km of 30 +/- 7 microM and a Vmax of 198 +/- 13 nmoles ethanolamine formed per hour per mg protein homogenate was obtained.