N-ras oncogene–induced gene expression in human hematopoietic progenitor cells: Upregulation of p16INK4a and p21CIP1/WAF1 correlates with myeloid differentiation

Objectives Mutations in ras oncogenes occur at high frequency in acute myeloid leukemia and myelodysplastic syndromes; however, the role of ras genes in leukemogenesis has not been clearly defined. Our previous studies have shown that expression of mutant N-ras (N-rasG13R, G to C transversion) in human hematopoietic progenitor cells (HPC) promotes myeloid differentiation and proliferation both in vitro and in a NOD/SCID mouse model. In the present study, we performed expression profiling to identify the transcriptome induced by N-rasG13R in human HPC, and analyzed the effect of mutant N-ras in sorted specific subpopulations of HPC. Methods cDNA microarray analysis was performed on cord blood CD34+ cells transduced with a retrovirus containing GFP alone or in combination with mutant N-ras. Transduced cells were also sorted into factorial subpopulations according to CD34 and transgene expression, and analyzed in suspension or semi-solid methylcellulose culture. Results Among a variety of changes, including upregulation of cytokine genes, we found that N-rasG13R induced expression of the cyclin-dependent kinase inhibitors p16INK4a and p21CIP1/WAF1. Analysis by RT-PCR revealed that increased p16INK4a and p21CIP1/WAF1 occurred in the most primitive, CD34+/Ras+ population but not in the more mature CD34−/Ras+ cells or in the CD34+/Ras− cells. Moreover, N-rasG13R inhibited the proliferation of the primitive CD34+/Ras+ cells, both in liquid culture and in colony assays. This growth suppression correlated with an increased proportion of myelomonocytic colonies and a decrease of erythroid colonies. In contrast, the growth of CD34−/Ras+ cells and CD34+/Ras− HPC was not inhibited. Conclusions These findings demonstrated the mutant N-ras induced transcriptome, and that this is associated with HPC gowth suppression / myelomonocytic differentiation, and identify upregulation of cyclin inhibitors as key events in this process. The results indicate that ras mutation alone is not sufficient to induce leukemogenesis; collaborative secondary event(s) are involved in the process.