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Oncogenic c-kit transcript is a target for binase
Vladimir A Mitkevich1, Irina Y Petrushanko, Olga V Kretova
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
Abstract:
Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase--RNase from Bacillus intermedius--on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.
Insights
Binase, an RNase, selectively kills acute myelogenous leukemia cells expressing mutated c-Kit by degrading its mRNA. Inhibiting the c-Kit protein reduces this sensitivity, highlighting mRNA as the key target.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Mutations in the c-Kit receptor tyrosine kinase are frequent in acute myelogenous leukemia (AML).
- The N822K point mutation in c-Kit confers interleukin-3 independent growth to myeloid progenitor cells (FDC-P1).
Purpose of the Study:
- To investigate the selective cytotoxic effects of binase (an RNase from Bacillus intermedius) on FDC-P1-N822K cells.
- To determine if the expression of the c-Kit oncogene is responsible for the observed sensitivity to binase.
- To explore the potential of targeting both the oncogenic protein and its mRNA for cancer therapy.
Main Methods:
- Culturing FDC-P1-N822K cells, which express the N822K c-Kit mutation.
- Treating cells with binase and imatinib (a c-Kit inhibitor).
- Assessing cell viability and apoptosis.
- Quantifying cellular RNA and c-Kit mRNA levels using quantitative RT-PCR.
- Utilizing green fluorescent protein as a marker for c-Kit oncoprotein expression.
Main Results:
- Binase exhibits selective cytotoxicity towards FDC-P1-N822K cells.
- Inhibition of the mutated c-Kit protein with imatinib significantly reduces cell sensitivity to binase-induced apoptosis.
- Binase treatment leads to a 41% reduction in c-Kit mRNA levels in treated cells.
- The elimination of c-Kit is identified as the critical factor for binase's selective cytotoxicity.
Conclusions:
- The activated mutant c-Kit transcript is a direct target for the toxic action of binase.
- Targeting both the oncogenic protein and its corresponding mRNA represents a promising strategy for eliminating malignant cells in AML.
