Abstract | BACKGROUND: METHODS: All possible point mutations in the DNA triplet codon that could result in amino acid substitutions at Thr670 (Thr670Arg, Thr670Ile, Thr670Lys, Thr670Ala, Thr670Ser, Thr670Pro) were introduced by site-specific mutagenesis of the complementary DNA for a constitutively active, imatinib-sensitive form of the KIT receptor, Delta559/KIT. The resulting mutant KIT proteins were transiently expressed in COS1 African green monkey kidney cells grown with and without imatinib, and cell extracts were analyzed for KIT activation by immunoprecipitation and immunoblotting to determine autophosphorylation levels. We also performed molecular modeling to estimate the relative affinities of wild-type (Thr670) KIT and the KIT mutants for ATP and imatinib. RESULTS: Like the parental strain, Thr670Ala, Thr670Ser, and Thr670Lys mutants were inhibited by 5 microM imatinib, but in comparison, they were only weakly active and Thr670Pro and Thr670Arg were not active at all. Only the Thr670Ile mutant was fully active (autophosphorylated) and resistant to imatinib. These findings were consistent with computer modeling predictions that ranked these mutants Thr - Ile > Ala, Ser > Lys >> Pro according to their affinity for ATP but Thr > Ala, Ser > Lys >Pro - Arg - Ile according to their affinity for imatinib. CONCLUSIONS: This combination of in vitro and molecular modeling analyses shows why, among all possible amino acid substitutions at position 670 of KIT, only Ile is naturally selected as a resistance mutant in imatinib-treated GIST patients.
|
Authors | Tiziana Negri, Giovanni Maria Pavan, Emanuela Virdis, Angela Greco, Maurizio Fermeglia, Marco Sandri, Sabrina Pricl, Marco A Pierotti, Silvana Pilotti, Elena Tamborini |
Journal | Journal of the National Cancer Institute
(J Natl Cancer Inst)
Vol. 101
Issue 3
Pg. 194-204
(Feb 04 2009)
ISSN: 1460-2105 [Electronic] United States |
PMID | 19176456
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
|
Chemical References |
- Antineoplastic Agents
- Benzamides
- Piperazines
- Protein Kinase Inhibitors
- Pyrimidines
- Threonine
- Imatinib Mesylate
- Proto-Oncogene Proteins c-kit
|
Topics |
- Antineoplastic Agents
(pharmacology)
- Benzamides
- Computer Simulation
- Drug Resistance, Neoplasm
- Gastrointestinal Stromal Tumors
(genetics)
- Humans
- Hypereosinophilic Syndrome
(genetics)
- Imatinib Mesylate
- Immunoblotting
- Immunoprecipitation
- In Vitro Techniques
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive
(genetics)
- Models, Molecular
- Mutation, Missense
- Piperazines
(pharmacology)
- Point Mutation
- Protein Kinase Inhibitors
(pharmacology)
- Proto-Oncogene Proteins c-kit
(genetics)
- Pyrimidines
(pharmacology)
- Reproducibility of Results
- Research Design
- Threonine
|