title: 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. We have shown recently that decreased cAMP-responsive element (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 cAMP response element-binding protein 1 (pCREB-1) and CRE-binding. Consistent with the autoregulation 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 the restoration of CRE-binding, CRE-reporter activity, and CREB-1 and RFC mRNA levels. Hence, the protein kinase A pathway was examined using various agents that augment intracellular cAMP levels, including cholera toxin, an upstream agonist that renders stimulatory G-proteins (Galphas) constitutively active. Treatment of antifolate-resistant cells with these agents resulted in the 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 Galphas or their coupled receptors, resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes.
authors.affiliation: Department of Biology, the Technion-Israel Institute of Technology, Haifa, 32000, Israel.
authors.forename: Lilah
authors.initials: L
authors.lastname: Rothem
authors.forename: Michal
authors.initials: M
authors.lastname: Stark
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein; D003520:Cyclophosphamide / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D015458:Leukemia, T-Cell; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000235:genetics*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection
chemical_list: C115163:CREB1 protein, human; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014157:Transcription Factors; D005576:Colforsin; D003994:Bucladesine; D003520:Cyclophosphamide; D015056:1-Methyl-3-isobutylxanthine