טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRothem Lilah
SubjectCharacterization of the Moleculr Mechanisms of Resistance
to Novel Antifolate Anticancer Drugs
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf


Abstract

The human reduced folate carrier (hRFC) is the dominant transporter mediating the uptake of reduced folates and antifolate anticancer drugs. Defective antifolate uptake due to inactivating hRFC mutations is an established mechanism of drug resistance in various tumor cells. However, while antifolate transport is frequently impaired, either no or only a single hRFC allele is inactivated, suggesting that additional mechanism(s) of resistance are operative. Here we describe a novel mechanism of antifolate resistance based on transcriptional silencing via loss of transcription factor binding to the hRFC promoter in the absence of transporter mutations. Antifolate-resistant human leukemia cells established by stepwise drug selection showed an abolished antifolate uptake due to the lack of RFC expression. The hRFC promoter contains an upstream GC-box and a downstream cAMP-response element (CRE)/AP1-like element. Electrophoretic mobility shift assays and oligonucleotide competition revealed the loss of nuclear factor binding to CRE and GC-box in these cell lines. Antibody-mediated supershift analysis showed a profound decrease in the binding of CRE-binding protein 1 (CREB-1) to CRE, and specificity protein 1 (Sp1) to the GC-box. Transient transfections into these cell lines demonstrated a nearly complete loss of GC-box-dependent, and ~ 70% decrease in CRE-driven reporter gene activity. These cell lines had undetectable CREB-1 expression, ~3-fold decreased ATF-1 levels, parental Sp1 levels, and 3-fold increased levels of the short Sp3 isoforms, recently shown to repress hRFC expression. These results provide the first demonstration of a novel mechanism of antifolate resistance based on abolished drug transport due to the loss of transcription factor expression and binding to the hRFC promoter.