The thesis is devoted to study of the cell-specific role of cis- and trans-acting factor in regulation of GSTP1 gene transcription in three malignant cell lines (breast cells Hbl-100, melanoma cells Me45 and choriocarcinoma cells BeWo), which possess different levels of GSTP1 expression and identification of changes in transcriptional regulation associated with neoplastic transformation of trophoblast cells. ARE and NF-κB sites were identified as positive and CRE and NF-κB-like site as negative regulatory elements. Proteins interacting with these sites were also identified. We demonstrated that CRE and NF-κB sites are responsible for cell-specific regulation of the GSTP1 transcription. The mode of ERβ and Fos interaction together with Jun binding mediates cell-specific influence of CRE and subunit composition of NF-κB is responsible for cell-specific action of NF-κB site. It was indicated that down-regulation of GSTP1 during placental carcinogenesis occurs due to lack of Jun in CRE-protein complex. We also demonstrated that in cells with transcription factor-mediated GSTP1 repression promoter of this gene undergoes methylation.

Glutathione S-transferase P1 is a major phase II detoxification enzyme in most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. GSTP1 gene transcription is regulated by promoter methylation and by transcription factors. To elucidate the mechanisms responsible for the different levels of GSTP1 expression observed in Hbl-100 and BeWo cells we utilized truncated promoter constructs to compare the functional role of different promoter elements. We also identified transcription factors binding the responsive elements by electrophoretic mobility shift assay. The applied approaches provided the evidence that binding of transcription factors to ARE, CRE and NF-kB sites are responsible for the cell specific levels of GSTP1 expression in Hbl-100 and BeWo cells. It was also indicated that partial promoter methylation occurs in BeWo cells.

im. The human GSTP1 is a major enzyme of phase II detoxification in the most cell types. Aberrant expression of GSTP1 is associated with carcinogenesis and development of multidrug resistance. The GSTP1 gene expression is regulated at multiple levels including transcriptional, post-transcriptional and post-translational. We concentrated our attention on the transcriptional level of regulation. Methods. Transient transfection of Me45 melanoma cells with constructs containing the luciferase gene under the control of complete and truncated GSTP1 promoter was utilized to identify a role of different promoter regions in regulation of the gene transcription in Me45 cells. To identify the transcription factors, interacting with the GSTP1 promoter sites, the competitive EMSA and super shift assay were applied. Results. GSTP1 transcription in Me45 cells is positively regulated by binding NF-kB to the cognate site and ERb in complex with unknown protein to the ARE site; the complex of ERb with c-Fos negatively regulates the gene expression via CRE site. The interaction of c-Fos/ERb with GSTP1 CRE site and indirect interaction of ERb with GSTP1 ARE were identified. Conclusions. The positive regulation of the human GSTP1 gene in Me45 melanoma cells is exerted via NF-kB and ARE sites and the negative one via CRE site of the promoter. ERb is indirectly involved in the regulation of GSTP1 transcription. It is bound via c-Fos with CRE site and via unknown protein with ARE site.
Crosstalk between transcription factors in regulation of the human glutathione S-transferase P1 gene expression in Me45 melanoma cells / А.М. Slonchak, А. Cwieduk, J. Rzerzowska-Wolny, M.Yu. Obolenskaya // Біополімери і клітина. — 2009. — Т. 25, № 3. — С. 210–217. — Бібліогр.: 22 назв. — англ.