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Department of Biology, the Technion-Israel Institute of Technology, Haifa, Israel
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 (G
s) 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 Gs or their coupled receptors, resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes.
Address correspondence to: Dr. Yehuda G. Assaraf, Department of Biology, the Technion-Israel Institute of Technology, Haifa, 32000, Israel. E-mail: assaraf{at}tx.technion.ac.il
This article has been cited by other articles:
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  I. Ifergan, G. Jansen, and Y. G. Assaraf The Reduced Folate Carrier (RFC) Is Cytotoxic to Cells under Conditions of Severe Folate Deprivation: RFC AS A DOUBLE EDGED SWORD IN FOLATE HOMEOSTASIS J. Biol. Chem., July 25, 2008; 283(30): 20687 - 20695. [Abstract] [Full Text] [PDF]
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 Y. Kaufman, I. Ifergan, L. Rothem, G. Jansen, and Y. G. Assaraf Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss Blood, April 15, 2006; 107(8): 3288 - 3294. [Abstract] [Full Text] [PDF]
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M. Stark and Y. G. Assaraf Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier Blood, January 15, 2006; 107(2): 708 - 715. [Abstract] [Full Text] [PDF]
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 L. Rothem, B. Berman, M. Stark, G. Jansen, and Y. G. Assaraf The Reduced Folate Carrier Gene Is a Novel Selectable Marker for Recombinant Protein Overexpression Mol. Pharmacol., September 1, 2005; 68(3): 616 - 624. [Abstract] [Full Text] [PDF]
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