טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShafran Assaf
SubjectSingle Amino Acid Substitutions in the Multidrug Efflux
Transporter ABCG2 Confer High Level Resistance to
Various Antifolates
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf


Abstract

Overexpression of the ABC transporter ABCG2 has been shown to confer resistance to a variety of chemotherapeutic agents. sequence analysis of the ABCG2 cDNAs identified acquired mutations, G482. Introduction of the mutant G482 ABCG2 genes into drug sensitive cells resulted in altered ABCG2 substrate specificity and major shifts in the cross-resistance profiles.

Whereas the wild type (R482) ABCG2 transported MTX and conferred a substantial level of resistance to this antifolate upon a continuous 7 days drug exposure, the mutant G482 failed to confer resistance to MTX under these conditions.

 we here characterized the role of the G482 ABCG2 mutation on cellular resistance to various antifolates including MTX as compared to the wild type R482 counterpart and parental HEK293 cells. We find that cells with overexpression of the G482 ABCG2 display 120-fold to more than 6,250-fold resistance to antifolates upon a 4 hr exposure, relative to parental HEK293 cells; however, these cells lost much of this antifolate resistance upon a continuous 72 hr drug exposure. Furthermore, this antifolate resistance was completely reversed with Ko143, a potent and specific ABCG2 inhibitor. Consistently, these G482 ABCG2 overexpressing cells displayed a 2-fold decrease in the 4 hr transport of [3H]MTX relative to their parental cells. Furthermore, a marked decrease in the accumulation of MTX polyglutamates was found in G482- and R482 ABCG2 cells. These results establish that the G482 ABCG2 is a dominant gain of function mutation not only in regards to resistance to anthracyclines but also to antifolates.