The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, ga...The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, gain-of-function mutations alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the onset or progression of cancerous transformations. The most common mutation of c-Kit is the substitution of aspartic acid residue in position 816 to valine (D816V), which is majorly responsible for its ligand-independent constitutive activation, and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy is increasingly becoming a hot spot due to its specificity and low toxicity. As the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling mediated downstream molecular targets. Specifically, we created c-Kit active mutant form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced cellular mitogenic activity compared to wild-type c-Kit expressing cells independent of huSCF. To examine the molecular targets regulating tumorogenic proliferation, we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene expression profile by high throughput microarray technology. The levels of some of the relevant genes (PIK3CB, eIF4B, PRKCDBP, MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B, MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit. Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP kinase.展开更多
文摘The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, gain-of-function mutations alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the onset or progression of cancerous transformations. The most common mutation of c-Kit is the substitution of aspartic acid residue in position 816 to valine (D816V), which is majorly responsible for its ligand-independent constitutive activation, and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy is increasingly becoming a hot spot due to its specificity and low toxicity. As the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling mediated downstream molecular targets. Specifically, we created c-Kit active mutant form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced cellular mitogenic activity compared to wild-type c-Kit expressing cells independent of huSCF. To examine the molecular targets regulating tumorogenic proliferation, we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene expression profile by high throughput microarray technology. The levels of some of the relevant genes (PIK3CB, eIF4B, PRKCDBP, MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B, MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit. Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP kinase.
