title: N-ras oncogene-induced gene expression in human hematopoietic progenitor cells: upregulation of p16INK4a and p21CIP1/WAF1 correlates with myeloid differentiation.
abstract: OBJECTIVE
  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 p16(INK4a) and p21(CIP1/WAF1). Analysis by RT-PCR revealed that increased p16(INK4a) and p21(CIP1/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 growth 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.
authors.affiliation: Children's Cancer Institute Australia, Randwick, Sydney, Australia.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Shen
authors.forename: Toby
authors.initials: T
authors.lastname: Passioura
authors.forename: Geoff
authors.initials: G
authors.lastname: Symonds
authors.forename: Alla
authors.initials: A
authors.lastname: Dolnikov
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis* / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis* / Q000235:genetics; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020125:Mutation, Missense; D009190:Myelodysplastic Syndromes / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D023461:Myeloid Progenitor Cells / Q000378:metabolism* / Q000473:pathology; D015854:Up-Regulation / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016207:Cytokines