The intricate role of Smads in the regulation of FPGS gene expression

Abstract

Antifolates are important components of treatment regimens of hematological malignancies and solid tumors. Following cellular uptake, antifolates undergo polyglutamylation, catalyzed by the enzyme folylpoly-γ-glutamate synthetase (FPGS), which is crucial for their intracellular retention and cytotoxic activity. Hence, loss of FPGS activity results in diminished antifolate polyglutamylation and antifolate resistance. Aberrant TGF-β/Smad signaling is thought to be a major contributor to leukemogenesis, as members of this pathway are negative regulators of hematopoiesis. Recently we characterized the human antifolate resistant leukemia cell line MTA C-3 which lost 97% of its cellular FPGS activity and harbors a heterozygous point mutation in exon12 of FPGS. This resulted in the loss of 99% of the expression of the wild type FPGS allele, while the expression of the mutant allele was retained. Research into the molecular mechanism underlying the selective silencing of the wild type allele in these cells established exon12 of FPGS as an intragenic transcriptional regulator, with the ability to drive transcription in vitro . We further showed that exon12 can be occupied by many transcription factors and chromatin remodeling proteins (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo and that the expression of FPGS is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12, both in acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. Here we further demonstrate the possible roles of Smads in the regulation of FPGS gene expression. We found that treatment of MTA C-3 cells with 5-aza-deoxycytidine and trichostatin A results in repression of the mutant allele as well as in decreased expression of FPGS in drug sensitive parental CCRF-CEM cells. This repression correlated with an increased expression of Smad3 and the inhibitory Smad7. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.