RESUMEN
Sphingosine 1-phosphate (S1P) is known to regulate cell proliferation, apoptosis, and motility. Recently, we have reported that S1P and its analogue dihydro-S1P (DHS1P) promote proliferation of rat cultured mesangial cells. To investigate the signaling mechanisms underlying S1P- and DHS1P-induced mesangial cell proliferation, we performed cDNA microarray analysis of gene expression during mesangial cell proliferation. In terms of the overall pattern, gene expression waves induced by S1P and DHS1P were similar to those induced by a potent mesangial mitogen platelet-derived growth factor (PDGF), whereas we found several genes, such as two growth factors, connective tissue growth factor (CTGF) and heparin-binding EGF-like growth factor (HB-EGF), which were induced by the sphingolipids, but not by PDGF. Cluster analysis also identified calcium-dependent molecules highly expressed in DHS1P-stimulated cells compared to S1P-stimulated cells. Calcium mobilization analysis showed that DHS1P had higher magnitudes of intracellular calcium mobilization than S1P, suggesting that S1P and DHS1P differentially regulate intracellular calcium mobilization, possibly leading to different gene expression in mesangial cells. The large-scale monitoring of gene expression performed here allows us to identify S1P-induced transcriptional properties during mesangial cell proliferation.
Asunto(s)
Mesangio Glomerular/metabolismo , Lisofosfolípidos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Transcripción Genética , Animales , Calcio/análisis , División Celular , Perfilación de la Expresión Génica , Mesangio Glomerular/química , Mesangio Glomerular/efectos de los fármacos , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
The acute lymphoblastic leukemia cell line CCRF-CEM is sensitive to Ara-C and undergoes apoptosis. In contrast, the chronic myelogenous leukemia (CML) cell line K562 is highly resistant to Ara-C, which causes the cells to differentiate into erythrocytes before undergoing apoptosis. We used cDNA microarrays to monitor the alterations in gene expression in these two cell lines under conditions leading to apoptosis or differentiation. Ara-C-treated CCRF-CEM cells were characterized by a cluster of down-regulated chaperone genes, whereas Ara-C-treated K562 cells were characterized by a cluster of up-regulated hemoglobin genes. In K562 cells, Ara-C treatment induced significant down-regulation of the asparagine synthetase gene, which is involved in resistance to L-asparaginase. Sequential treatment with Ara-C and L-asparaginase had a synergistic effect on the inhibition of K562 cell growth, and combination therapy with these two anticancer agents may prove effective in the treatment of CML, which cannot be cured by either drug alone.
Asunto(s)
Citarabina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Células K562/efectos de los fármacos , Células K562/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
BACKGROUND: The bioactive sphingolipid sphingosine 1-phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet-derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P-induced proliferation of mesangial cells is poorly known. RESULTS: We examined the signalling mechanisms by which S1P and dihydro-S1P (DHS1P), another S1P receptor agonist, induce mesangial cell proliferation. We first observed that exogenous S1P/DHS1P had additive effects on the PDGF-promoted proliferation of mesangial cells. Treatment of mesangial cells with pertussis toxin almost completely inhibited S1P- and DHS1P-induced, and slightly inhibited PDGF-induced cell proliferation. Additionally, the ERK kinase inhibitor PD98059 partially blocked the proliferation of mesangial cells induced by all these ligands. N,N-dimethylsphingosine, a competitive inhibitor of SPHK, reduced PDGF-induced mesangial cell proliferation, whereas over-expression of SPHK promoted it. We also revealed that PDGF induces SPHK mRNA expression and SPHK activity, suggesting that SPHK, which links the PDGF to the S1P signalling cascade, is, at least in part, involved in PDGF-induced mesangial cell proliferation. Moreover, we found that extracellular S1P stimulates two S1P receptors, EDG3 and EDG5, which leads to cell proliferation and survival. CONCLUSIONS: The data show that S1P-induced mesangial cell proliferation is mediated by EDG-dependent and -independent signalling pathways. S1P may cooperate with PDGF to increase the proliferation of mesangial cells during pathophysiological processes.