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01 July 2006

Up-regulation of p21WAF1 expression is mediated by Sp1/Sp3 transcription factors in TGFbeta1-arrested malignant B cells.

Daniel Tvrdík, Pavel Dundr, Ctibor Povýsil, Robert Pytlík

Med Sci Monit 2006; 12(7): BR227-234 :: ID: 452194

Abstract

BACKGROUND: TGFbeta1 has a profound effect on the growth of various mammalian cell types, including B lymphocytes. The inhibitory action of TGFbeta1 is mediated by deactivation of the cell cycle machinery. Several feedback-sensitive pathways determine whether the cells are stopped in G1 phase or allowed to leave G1 phase and enter S phase. Cell cycle-associated molecules, e.g. cyclin-dependent kinase inhibitors (CKIs), may become targets for the inhibitory signaling pathways induced by TGFbeta1. MATERIAL/METHODS: Our experimental DoHH2 cell line model was derived from a patient with malignant non-Hodgkin's lymphoma of follicular origin. The effect of TGFbeta1 on cell cycle progression was studied by flow cytometry. We examined the effect of TGFbeta1 on the expression of p21WAF1 by immunoblotting and RT-PCR. The binding activity of transcription factors to the p21 gene promoter was determined by gel mobility shift assay (GMSA). RESULTS: Our results showed that TGFbeta1 treatment increased the number of cells arrested in G0/G1 phase compared with untreated control cells. Moreover, we found that p21WAF1 expression was significantly up-regulated on the protein level after TGFbeta1 treatment. Similarly to the protein level, the expression of p21 mRNA was increased in TGFbeta1-treated cells. We further examined the binding activity of the Sp family of transcription factors to examine their role in p21WAF1 up-regulation. CONCLUSIONS: The results indicated that p21WAF1 over-expression in TGFbeta1-arrested malignant B cells is mediated by binding of Sp1/Sp3 transcription factors to the (-92/-71), (-77/-58), and (-65/-45) elements of the promoter region of the p21 gene.

Keywords: Base Sequence, B-Lymphocytes - metabolism, Blotting, Western, Cell Cycle - drug effects, Cell Line, Cyclin-Dependent Kinase Inhibitor p21 - physiology, DNA Primers, Electrophoretic Mobility Shift Assay, Lymphoma, B-Cell - pathology, Reverse Transcriptase Polymerase Chain Reaction, Sp1 Transcription Factor - physiology, Sp3 Transcription Factor - physiology, Transforming Growth Factor beta - pharmacology

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750