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  • Concerning the molecular mechanisms underlying transcription

    2022-06-23

    Concerning the molecular mechanisms underlying transcriptional regulation of the GSTP1-1 gene expression we and others have characterized structural and functional properties of the human GSTP1-1 promoter. GSTP1-1 gene promoter activity is driven by a TATA-box [16] as well as Spl [16], [17], AP-1 [18] and NF-κB [19] binding sites. More recently, we demonstrated that the transcription factor GATA-1 binds to a GATA sequence located in −1211/−1208 of the GSTP1-1 gene promoter. Indeed, we showed a correlation between GATA-1 binding activity as well as GSTP1-1 mRNA and protein expression during erythroid- and megakaryocytic-induced K562 cell differentiation [20]. K562 cell line is a well-established model system for the study of spontaneous as well as induced differentiation towards erythrocytic, granulocytic, and monocytic lineages [21]. An alternative approach to classical anti-cancer therapy consists in the induction of cell differentiation using low doses of agents devoid of cytotoxic effect [22], [23], [24], [25]. However, it is important to further investigate if this type of approach to treatment may increase cancer cell resistance following cell detoxification by GSTs. In order to determine whether GSTP1-1 gene expression could be linked to induction of erythroid differentiation by pharmacological agents, we investigated the effect of hemin, a canonical erythroid differentiating agent of K562 cells. Hemin exerts a profound effect on cell maturation and promotes foetal hemoglobin synthesis in erythroid progenitors cultured in vitro [26] and serves as a therapeutic agent in human porphyria treatment [27]. Here we establish a connection between hemoglobin production, GSTP1-1 mRNA and protein expression in hemin-treated K562 cells. We studied the response of transcription factors involved in the regulation of GSTP1-1 expression such as GATA-1 [20], NF-κB [19] and AP-1 [18]. Our findings suggest that GSTP1-1 overexpression in hemin-induced differentiation of K562 BMS-626529 is not primarily due to a transcriptional upregulation but rather to a stabilization of the corresponding mRNA. In conclusion, we demonstrate the existence of post-transcriptional mechanisms regulating GSTP1-1 gene expression during hemin-induced differentiation of K562 cells.
    Materials and methods
    Results
    Discussion During the differentiation of erythroid cells, a maturation takes place leading to the elimination of organelles including the nucleus. In order to assure the regulated expression of vital proteins, mRNA stabilization is known to contribute to maintain physiological functions of the red blood cells. We previously showed that induction of erythroid differentiation by aclarubicin and doxorubicin leads to an increase in GSTP1-1 expression. In contrast, TPA, a megakaryocytic pathway inducer, inhibited constitutive GSTP1-1 expression at both mRNA and protein levels [20]. For this work, we used the canonical erythroid differentiating agent hemin. The molecular effect of hemin at the transcriptional level was previously described by Partington and Patient using EMSA [30]. Indeed, GATA-1 DNA-binding activity to the α-globin gene promoter increased after induction of erythroid differentiation of K562 cells by 50μM hemin. Our results confirm partially these findings as we observe an initial increase in GATA binding activity after 24h of treatment. Our results obtained at a differentiating concentration of 30μM show that a prolonged treatment by hemin induces a reduction of GATA-1 mRNA expression as well as transcription factor binding activity to the GSTP1-1 GATA site located at −1208 relative to the transcriptional start site. Site specific differences between both α-globin and GSTP1-1 GATA sites as well as differential phosphorylation of the GATA-1 factor could provide elements to explain a differential affinity of the GATA transcription factor for the GSTP1-1 gene promoter. Nevertheless, other post-transcriptional regulatory mechanisms should also be taken into consideration for a better understanding of the regulated expression of GSTP1-1.