Étude de l'effet de la cytokine TNFa sur la régulation de l'expression de la [gamma]-glutamyl-transférase humaine

Abstract : [gamma]-glutamyltransferase (GGT) is an enzyme that catalyzes the hydrolysis of glutathione, the major cellular antioxidant. This enzyme is overexpressed in many cancers (for example in leukemia) and belongs to a multigene family of at least thirteen genes. The GGT gene I, which encodes the active protein, is transcribed into three mRNAs (A, B and C), all showing the same open reading frame but diffeiring in their 5' untranslated region. Besides the involvement of GGT in the development of cancers, it is also involved in the acquisition of resistance to anticancer treatments and in the synthesis of leukotrienes. During this thesis, we were interested in the transcriptional mechanisms that regulate GGT gene I expression. Specifically, we studied the regulation of GGT after treatment of K562 cells with the tumor promoter TPA, and the pro-inflammatory cytokine, TNF[alpha]. Both substances activate the transcription factors AP-1 and NF-[kappa]B, which are known to regulate genes involved in the development of chemoresistance. Our results show that the two GGT promoters B and C and the three mRNA A, B and C, as well as GGT protein and GGT enzymatic activity are induced by TNF[alpha], but not by TPA in K562 cells, which are originated from a chronic myeloid leukemia. Since the induction of the GGT promoter C by TNF[alpha] is reduced with curcumin, a natural chemopreventif agent used as an inhibitor of the NF-[kappa]B signaling pathway, we supposed that the transcription factor NF-[kappa]B is involved in the regulation of the GGT promoter C in K562 cells. To prove our hypothesis, we used siRNA targeting specifically the two main subunits of NF-[kappa]B, p50 and p65, and indeed the induction of the GGT promoter C by TNFa is inhibited by these siRNA. Co-transfection assays with proteins of the NF-[kappa]B signaling pathway, TRAF2, TRADD, I[kappa] B[alpha] and p65, further confirm our hypothesis that the GGT promoter C is regulated via the NF-[kappa]B signaling pathway in K562 cells . Mutation experiments then identified the site on the DNA that is responsible for the induction of the GGT promoter C by TNFa in K562 cells. This site, located at -123 to -111 base pairs compared to the transcriptional start site, is able to bind at least the transcription factor p50 NF-[kappa]B. Near this site, we have identified another functional site, localized at - 73 - 92 base pairs compared to the transcriptional start site, which binds the transcription factor Sp1. Analysis by chromatin immunoprecipitation (or ChIP) confirmed the binding of the transcription factors NF-[kappa]B and Sp1 on the GGT promoter C and show that RNA polymerase II is also recruited at this specific location of the GGT promoter C. In addition, the analysis showed that treatment with TNFa induced binding of the transcription factors p50 NF-[kappa]B and Sp1, compared to untreated cells, and strongly increases the recruitment of RNA polymerase II to this location of the GGT promoter C, thus explaining the observed induction of the GGT promoter C after treatment with TNF[alpha]. In conclusion, the induction of GGT by TNF[alpha] may have several important biological significance, namely to increase resistance to anticancer drugs, to defend against oxidative stress or to induce leukotriene synthesis and thus to mediate inflammation.
Complete list of metadatas

Cited literature [93 references]  Display  Hide  Download

https://hal.univ-lorraine.fr/tel-01748440
Contributor : Thèses Ul <>
Submitted on : Thursday, March 29, 2018 - 11:35:06 AM
Last modification on : Monday, April 16, 2018 - 10:40:18 AM
Long-term archiving on: Friday, September 14, 2018 - 8:45:14 AM

File

SCD_T_2008_0054_REUTER.pdf
Files produced by the author(s)

Identifiers

  • HAL Id : tel-01748440, version 1

Collections

Citation

Simone Reuter. Étude de l'effet de la cytokine TNFa sur la régulation de l'expression de la [gamma]-glutamyl-transférase humaine. Sciences agricoles. Université Henri Poincaré - Nancy 1, 2008. Français. ⟨NNT : 2008NAN10054⟩. ⟨tel-01748440⟩

Share

Metrics

Record views

88

Files downloads

26