Abnormality of c-kit oncoprotein in certain patients with chronic myelogenous leukemia--potential clinical significance.
Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome and bcr/abl gene rearrangement which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate the biological properties of KIT in CML leukemogenesis, we performed analysis of alterations of the c-kit gene and functional analysis of altered KIT proteins. Gene alterations in the c-kit juxtamembrane domain of 80 CML cases were analyzed by reverse transcriptase and polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT --> AAG, Asn --> Lys), and six cases had the same base abnormality at codon 541 (ATG --> CTG, Met --> Leu) in the juxtamembrane domain. Because the change from Met to Leu at codon 541 was a conservative one which was also observed in the normal population and normal tissues of CML patients, it probably represents a polymorphic variation. Although samples of hair roots and leukemic cells from the chronic phase of one CML patient showed no abnormality, an abnormality at codon 541 (ATG --> CTG, Met --> Leu) was found only at blastic crisis (BC) of this case. In the case with the abnormality at codon 564, the mutation was detected only in a sample of leukemic cells collected at BC. To examine the biological consequence and biological significance of these abnormalities, murine KIT(L540) and KIT(K563) expression vectors were introduced into interleukin-3 (IL-3)-dependent murine Ba/F3 cells to study their state of tyrosine phosphorylation and their growth rate. Ba/F3 cells expressing KIT(WT), KIT(L540) and KIT(K563) showed dose-dependent tyrosine phosphorylation after treatment with increasing concentrations of recombinant mouse stem cell factor (rmSCF). The cells expressing KIT(L540) and KIT(K563) were found to have greater tyrosine phosphorylation than cells expressing KIT(WT) at 0.1 and 1.0 ng/ml of rmSCF. The Ba/F3 cells expressing KIT(K563) proliferated in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The Ba/F3 cells expressing KIT(L540)showed a relatively higher proliferative response to 0.1 ng/ml of rmSCF than the response of cells expressing KIT(WT). These mutations and in vitro functional analyses raise the possibility that the KIT abnormalities influence the white blood cell counts (P < 0.05) and survival (P < 0.04) of CML patients.
Inokuchi
K
K
Division of Hematology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan.
Yamaguchi
H
H
Tarusawa
M
M
Futaki
M
M
Hanawa
H
H
Tanosaki
S
S
Dan
K
K
D019943:Amino Acid Substitution; D000818:Animals; D001752:Blast Crisis / Q000235:genetics / Q000473:pathology; D001853:Bone Marrow / Q000737:chemistry / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000235:genetics* / Q000473:pathology; D015465:Leukemia, Myeloid, Accelerated Phase / Q000097:blood / Q000235:genetics / Q000473:pathology; D015466:Leukemia, Myeloid, Chronic-Phase / Q000097:blood / Q000235:genetics / Q000473:pathology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics*; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D011499:Protein Processing, Post-Translational; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D003062:Codon; D004273:DNA, Neoplasm; D007377:Interleukin-3; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; D019009:Proto-Oncogene Proteins c-kit; D016044:Fusion Proteins, bcr-abl