טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKaufman Yotam
SubjectMolecular Mechanisms of Antifolate Resistance: Genetic
Alterations and Silencing of the hRFC Gene
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf


Abstract

We have studied the molecular basis of reduced folate carrier (RFC)-dependent antifolate drug resistance in human CCRF-CEM leukemia cells that were resistant to the antifolate PT523, a dihydrofolate reductase inhibitor.  We found that these cells had an impaired RFC-dependent transport activity, and harbored four clustered mutations (Leu143Pro, Ala147Val, Arg148Gly, and Gln150amber) on a single hRFC allele, the last of which was a nonsense mutation.  These mutations originated from a relatively small population of cells that became predominant as the selection became more stringent.  Hence, this constitutes the first demonstration that selection of an advantageous clone from the entire population plays an important role in the acquisition of resistance to novel antifolates.  The resistant cells also had decreased levels of RFC protein and mRNA in comparison to WT cells.  We found that multiple antifolate-resistant sublines had a ~50% decrease in RFC gene copy number when compared to parental CEM cells.  We also found that three antifolate-resistant leukemia sublines (CEM GW70, EDXR2, and AMTR5) displayed a hRFC promoter methylation pattern that was different from that present in parental CEM cells.  Promoter methylation and allele loss could be factors that contribute to decreased levels of RFC protein and transport activity.  In a large screening clinical study of ~250 leukemia patients, we found that three acute lymphoblastic leukemia (ALL) patients harbored heterozygous hRFC mutations, rendering this mechanism of antifolate-resistance extremely rare in human ALL patients.  In a study of 42 ALL patients, none were found with a methylated RFC promoter.