Shen Sylvie, Passioura Toby, Symonds Geoff, Dolnikov Alla
Children's Cancer Institute Australia, Randwick, Sydney, Australia.
Exp Hematol. 2007 Jun;35(6):908-19. doi: 10.1016/j.exphem.2007.02.011.
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.
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.
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.
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.
Ras癌基因的突变在急性髓系白血病和骨髓增生异常综合征中高频发生；然而，Ras基因在白血病发生中的作用尚未明确界定。我们之前的研究表明，在人类造血祖细胞（HPC）中突变型N-Ras（N-RasG13R，G到C的颠换）的表达在体外和NOD/SCID小鼠模型中均促进髓系分化和增殖。在本研究中，我们进行了表达谱分析以鉴定N-RasG13R在人类HPC中诱导的转录组，并分析突变型N-Ras在分选的特定HPC亚群中的作用。
对用单独携带绿色荧光蛋白（GFP）或与突变型N-Ras联合的逆转录病毒转导的脐血CD34(+)细胞进行cDNA微阵列分析。转导的细胞还根据CD34和转基因表达被分选成分解亚群，并在悬浮或半固体甲基纤维素培养中进行分析。
在包括细胞因子基因上调在内的多种变化中，我们发现N-RasG13R诱导细胞周期蛋白依赖性激酶抑制剂p16(INK4a)和p21(CIP1/WAF1)的表达。逆转录聚合酶链反应（RT-PCR）分析显示，p16(INK4a)和p21(CIP1/WAF1)的增加发生在最原始的CD34(+)/Ras(+)群体中，而在更成熟的CD34(-)/Ras(+)细胞或CD34(+)/Ras(-)细胞中未出现。此外，N-RasG13R在液体培养和集落测定中均抑制原始CD34(+)/Ras(+)细胞的增殖。这种生长抑制与髓单核细胞集落比例增加和红系集落减少相关。相比之下，CD34(-)/Ras(+)细胞和CD34(+)/Ras(-) HPC的生长未受抑制。
这些发现证明了突变型N-Ras诱导的转录组，且这与HPC生长抑制/髓单核细胞分化相关，并确定细胞周期蛋白抑制剂的上调是该过程中的关键事件。结果表明，单独的Ras突变不足以诱导白血病发生；该过程涉及协同的继发事件。
