Received for publication June 24, 2004.
Revised August 27, 2004.
Accepted for publication August 27, 2004.

Impaired CREB-1 Phosphorylation in Antifolate-Resistant Cell Lines with Down-Regulation of the Reduced Folate Carrier Gene

Abstract

The human reduced folate carrier (hRFC) is the dominant transporter for the uptake of antifolates used in cancer chemotherapy. Recently we have shown that decreased CRE-dependent transcription contributes to the loss of hRFC gene expression in multiple antifolate-resistant cell lines. This was associated with markedly decreased levels of phosphorylated CREB-1 (pCREB-1) and CRE-binding. Consistent with the auto-regulation of CREB-1 gene expression by pCREB-1, prominently decreased CREB-1 mRNA levels were observed in antifolate-resistant cells. We therefore explored the possibility that these cells were defective in CREB-1 phosphorylation, thereby resulting in down-regulation of some cAMP-responsive genes. Two-dimensional gel electrophoresis revealed that CREB-1 and its phosphoisoforms were markedly decreased in these cells. Treatment with forskolin, an activator of adenylyl cyclase, restored both CREB-1 and pCREB-1 levels; this resulted in restoration of CRE-binding, CRE-reporter activity, as well as CREB-1 and RFC mRNA levels. Hence, the protein kinase A (PKA) pathway was examined using various agents that augment intracellular cAMP levels including cholera toxin, an upstream agonist that renders stimulatory G-proteins (Gs) constitutively active. Treatment of antifolate-resistant cells with these agents resulted in restoration of pCREB-1 levels and CRE-reporter activity. Furthermore, transient transfection with a constitutively transcriptionally active VP16-CREB-1 that does not require phosphorylation for its activity resulted in restoration of CREB mRNA levels but not pCREB-1 levels. This is the first demonstration that resistance to various antifolates may potentially be associated with impaired activity of Gs or their coupled receptors resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP responsive genes.

Key words: Gs family, cAMP, CREB, DNA binding sites, Resistance