RESUMO
We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/fisiologia , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Vimblastina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Elementos Facilitadores Genéticos/efeitos dos fármacos , Células HT29 , Humanos , Proteínas I-kappa B/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , NF-kappa B/genética , Nocodazol/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Ativação Transcricional/efeitos dos fármacos , Moduladores de Tubulina , Regulação para CimaRESUMO
We investigated the expression of c-myc in HT29-D4, HBL100 and Caco-2 cells treated with microtubule stabilising (paclitaxel) or depolymerising agents (vinblastine, nocodazole). After induction by epidermal growth factor (EGF), c-myc expression decreased in HT29-D4 cells treated with all the antimicrotubule agents. In HBL100 and Caco-2, when microtubules were stabilised with paclitaxel, c-myc expression also decreased. In contrast, its expression increased after treatment with depolymerising agents. In both cell lines, we also observed that depolymerising agents alone induced c-myc expression whilst paclitaxel had no effect. This mRNA induction was confirmed at the protein level. In HT29-D4, no variation of c-myc expression was observed. Then, we showed that the increase of mRNA level was due to activation of gene transcription. These results indicate that modulation of c-myc expression varied depending on the cell lines used and the type of antimicrotubule agents. This work provides a potential link between the microtubular network and c-myc gene expression.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Genes myc/genética , Microtúbulos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Western Blotting , Células CACO-2/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29/metabolismo , Humanos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CYP1A1 is implicated in the bioactivation of procarcinogens such as polycyclic aromatic hydrocarbons. To date, no physiological compounds have been described as inducers of this gene. In this study, we have examined the role of serum in the regulation of CYP1A1 gene expression. After treatment of CaCo-2 cells with fetal bovine serum, CYP1A1 mRNA level increased to the same extent as that observed after 3-methylcholanthrene induction. The same effect was obtained after treatment with adult bovine or human serum. Evaluation of hnRNA level performed on CaCo-2 cells indicates that CYP1A1 induction by serum acts at least in part through transcriptional activation. Promoter region containing the XRE (1.56 kb) was tested in the CAT assay. No stimulation of this reporter gene was detected after serum treatment. These results demonstrate for the first time that physiological compound(s) contained in serum induces CYP1A1 gene expression by transcriptional activation independent of the AhR pathway.
Assuntos
Citocromo P-450 CYP1A1/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Adulto , Animais , Sequência de Bases , Células CACO-2 , Bovinos , Cloranfenicol O-Acetiltransferase/genética , Meios de Cultura , Citocromo P-450 CYP1A1/biossíntese , Primers do DNA/genética , Indução Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Metilcolantreno/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Carbamazepine (CBZ) is widely used in the treatment of epilepsy. The drug is principally metabolized by CYPs to 10, 11-epoxy carbamazepine (CBZ-E) but this metabolite more toxic than the parent drug, does possess anticonvulsant properties. In humans, CYP3A4, CYP2C8 and CYP1A2 have been shown to be implicated in CBZ biotransformation. Our purpose was to establish an experimental model to determine the interaction of CBZ with other antiepileptic drugs. We first identified the CYP isoforms that metabolized CBZ in rabbit. We used liver microsomes from rabbit treated with various compounds known to induce principally some CYPs subfamilies. Having tested all the compounds we demonstrated that only the animals treated with CYP3A inducers were able to metabolize CBZ strongly. The CBZ biotransformation was inhibited by anti CYP3A antibodies. All the CYP3A subfamily substrates specifically decrease CBZ-E formation. In our experiment we did not observe any inhibition with CYP2C substrate. These data provide evidence that in rabbit the CYP3A subfamily is primarily involved in CBZ metabolism. Using this model we investigated the interaction of CBZ with phenobarbital, phenytoin, ethosuccimide, primidone, progabide, vigabatrin and lamotrigine.
