טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMichal Stark
SubjectTranscriptional Silencing of the Reduced Folate Carrier as a
Novel Mechanism of Antifolate Resistance
DepartmentDepartment of Biology
Supervisor Full Professor Assaraf Yehuda
Full Thesis textFull thesis text - English Version


Abstract

Herein we explored the basis of the decrease/loss of gene expression of the major transporter for antifolate drugs, the human reduced folate carrier (hRFC), in tumor cell-lines. Assessment of the role of the various cis-acting elements in the hRFC promoter, revealed decreased binding either to GC-box, Mzf-1, AP-1, E-box or CRE oligonucleotides in ~50-80% of the cell-lines. Western blot analysis demonstrated decreased expression of all transcriptional activators except for Sp1 and Sp3. Consistent with the auto-regulation of CREB-1 gene expression by phosphorylated CREB-1 (pCREB-1), decreased CREB-1 mRNA levels were observed. We hence explored whether these cells were defective in CREB-1 phosphorylation. Two-dimensional gel electrophoresis revealed that CREB-1 and its phosphoisoforms were markedly decreased in these cells. Treatment with forskolin 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.

Despite the poor GC-box-binding, normal Sp1 levels were retained. We therefore explored the post-translational modifications that may disrupt Sp1 function. Phospho-affinity purification of nuclear proteins revealed that antifolate-resistant cells contained ~8-fold more phosphorylated Sp1 than their parental cells; treatment of these proteins with alkaline phosphatase restored GC-box binding. Hence, the protein kinase A (PKA) pathway was examined using various agents that augment intracellular cAMP. Treatment of antifolate-resistant cells with these agents resulted in restoration of pCREB-1 levels and CRE-reporter activity. However, the above treatment revealed no effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels may also 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 hRFC mRNA levels. This restoration was abolished by okadaic acid, a protein phosphatase 2A (PP2A) inhibitor. 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 was prevented by the co-presence of okadaic acid.

These findings provide the first evidence that: 1) Transcriptional silencing of the hRFC gene in various tumor cell-lines is a result of a simultaneous loss of function of multiple transcription factors. 2) Antifolate resistance may be associated with impaired activity of Gαs and/or their coupled receptors resulting in loss of CREB-1 phosphorylation and consequent down-regulation of cAMP-responsive genes. 3) Loss of hRFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A.