TY - JOUR
T1 - Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates
AU - Bram, Eran
AU - Ifergan, Ilan
AU - Shafran, Assaf
AU - Berman, Bluma
AU - Jansen, Gerrit
AU - Assaraf, Yehuda G
PY - 2006/12
Y1 - 2006/12
N2 - Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.
AB - Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.
KW - ATP Binding Cassette Transporter, Subfamily G, Member 2
KW - ATP-Binding Cassette Transporters/antagonists & inhibitors
KW - Antineoplastic Agents/chemistry
KW - Biological Transport/drug effects
KW - Cell Line
KW - Cell Proliferation/drug effects
KW - Cell Survival/drug effects
KW - Cisplatin/chemistry
KW - Dose-Response Relationship, Drug
KW - Doxorubicin/chemistry
KW - Drug Resistance, Neoplasm/genetics
KW - Fluorouracil/chemistry
KW - Folic Acid Antagonists/chemistry
KW - Heterocyclic Compounds, 3-Ring/metabolism
KW - Humans
KW - Indoles/chemistry
KW - Lipids/chemistry
KW - Molecular Structure
KW - Mutation/genetics
KW - Neoplasm Proteins/antagonists & inhibitors
KW - Paclitaxel/chemistry
KW - Pyrimethamine/analogs & derivatives
KW - Pyrimidines/chemistry
KW - Quinazolines/chemistry
KW - Transfection
KW - Trimetrexate/chemistry
U2 - 10.1007/s00280-006-0230-9
DO - 10.1007/s00280-006-0230-9
M3 - Article
C2 - 16612649
VL - 58
SP - 826
EP - 834
JO - Cancer Chemotherapy and Pharmacology
JF - Cancer Chemotherapy and Pharmacology
SN - 0344-5704
IS - 6
ER -