Oncogenes & tumor suppressors in acute myeloid leukemia

在我们实验室建立的髓系白血病模型中，我们最近一直在关注肿瘤抑制因子的作用。细胞周期蛋白依赖性激酶(CDK)抑制剂是一类在细胞周期调控中发挥重要作用的肿瘤抑制因子，在人类肿瘤中通过缺失、启动子甲基化或突变而失活。特别是，在很高比例的人类AML、MDS和编码细胞周期蛋白依赖性激酶(CDK)抑制物p15INK4b基因的所有启动子区域都被发现是高甲基化的。在小鼠中，编码cdk抑制因子p15INK4b(CDKN2B)的基因位于编码cdk抑制因子p16INK4a的CDKN2a近端的Chr 4上。此外，CDKN2a通过选择性剪接编码P19ARF，它是P53肿瘤抑制通路的正向调节因子。P15INK4b特别有趣，因为它在髓系细胞中的转录水平被分化和生长抑制细胞因子上调，并在单核细胞系的成熟细胞中高度表达。 在今年的一个研究领域，我们已经开始检查几个小鼠模型系统中的髓系白血病INK4基因转录的变化，试图将INK4a或b的失活与特定的癌基因诱导的白血病联系起来。第一种是由内源性c-myb诱导的肿瘤，通过将逆转录病毒插入基因组而激活，其他的是由携带c-myc基因的逆转录病毒诱导的。我们的数据显示，大多数Myb相关肿瘤不表达p15INK4b mRNA，而大多数Myc肿瘤确实表达这种RNA。Myb肿瘤中缺乏表达不是由于缺失或甲基化。进一步的体外研究表明，c-Myb本身可以抑制p15INK4b的表达。在Myc肿瘤中，Northern分析显示p15INK4b的mRNA和蛋白在大多数肿瘤中表达，而p16INK4a基因的mRNA很少表达。有趣的是，两个Myc肿瘤有异常的INK4转录本，被确定为p15Ex1与p16Ex2或p16Ex2或3的融合，这是由于包含EX1？用于p19ARF。因此，在c-myc不适当表达的情况下，我们的数据表明，CDKN4a基因位点的改变可能主要是由于细胞试图使p19ARF失活，从而使p53抑癌通路失活。 另一个研究领域涉及产生编码肿瘤抑制基因的等位基因缺失的小鼠，以便我们可以分析它们对髓系白血病的易感性。我们已经准备了p15INK4a基因敲除，并正在比较纯合子、杂合子和野生型表型对逆转录病毒诱导的白血病的易感性。

In models of myeloid leukemogenesis developed in our laboratory we have recently been focusing on the role of tumor suppressors. Cyclin dependent kinase (cdk) inhibitors are tumor suppresses that play an important role in cell cycle control and have been inactivated in human tumors either by deletion, promoter hypermethylation or by mutation. In particular, in a high proportion of human AML, MDS and ALL the promoter region of the gene encoding the cyclin dependent kinase (cdk) inhibitor p15INK4b has been found to be hypermethylated. In the mouse, the gene encoding the cdk inhibitor p15INK4b (Cdkn2b) is located on Chr 4 proximal to Cdkn2a which encodes the cdk inhibitor p16INK4a. Additionally, Cdkn2a encodes, through alternative splicing, p19ARF, a positive regulator of the p53 tumor suppressor pathway. p15INK4b is particularly interesting because it is upregulated at the transcription level in myeloid cells by differentiation and growth-inhibiting cytokines and is highly expressed in mature cells of the monocytic lineage. In one area of research this year we have began examining myeloid leukemias from several murine model systems for alterations in INK4 gene transcription in an attempt to correlate inactivation of INK4a or b with specific oncogene-induced leukemias. The first are neoplasms induced by endogenous c-myb, activated by insertion of retrovirus into the genome, while the others are induced by a retrovirus carrying the c-myc gene. Our data show that most Myb related tumors do not express p15INK4b mRNA, whereas most Myc tumors do express this RNA. A lack of expression in the Myb tumors is not due to deletion or methylation. Further studies in vitro have indicated that the c-Myb itself can repress the expression of p15INK4b. In the case of the Myc tumors, Northern analysis revealed that mRNA and protein for p15INK4b were expressed in a majority of tumors whereas the mRNA for the p16INK4a gene, was rarely expressed. Interestingly, two Myc tumors had aberrant INK4 transcripts that were determined to be fusions of p15Ex1 with p16Ex2 or 3 and this was due to deletion of a region encompassing Ex1? for p19ARF. Therefore, in the case of inappropriate c-myc expression, our data would suggest that alterations in the Cdkn4a locus may be primarily due to attempts by the cell to inactivate p19ARF and therefore the p53 tumor suppressor pathway. Another area of research involves the generation of mice with loss of alleles for the genes encoding tumor suppressors so that we can analyze them for susceptibility to myeloid leukemia. We have prepared a knockout of p15INK4a and are comparing the homozygous, heterozygous and wild-type phenotypes for susceptibility to retrovirus-induced leukemia.