Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier

Abstract

The reduced folate carrier (RFC) is the dominant influx transporter for antifolates. A major mechanism of antifolate resistance is loss of RFC (SLC19A1) gene expression due to decreased GC-box-dependent transcription. However, despite the poor GC-box binding in multiple antifolate-resistant cell lines, normal Sp1 levels were retained. Here we explored the post-translational modifications that may disrupt Sp1 function. Phosphoaffinity purification of nuclear proteins revealed that resistant cells contained approximately 8-fold more phosphorylated Sp1 than parental cells; treatment of nuclear proteins from these cells with alkaline phosphatase restored GC-box binding. As protein kinase A phosphorylates Sp1, resistant cells were treated with various cAMP-reactive agents, revealing no apparent effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels also may be affected by IBMX, resistant cells were treated with 8-pCPT-cGMP, resulting in the complete restoration of GC-box binding, luciferase reporter activity, and RFC mRNA levels. This restoration was abolished in the presence of the protein phosphatase 2A inhibitor (PP2A) okadaic acid. Importantly, whereas resistant cells showed multiple phosphorylated Sp1 forms barely detectable in parental cells, treatment with 8-pCPT-cGMP resulted in their elimination; this disappearance, however, was prevented by the copresence of okadaic acid. These findings provide the first evidence that loss of RFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A. © 2006 by The American Society of Hematology.