Oncogenes and tumor suppressors in murine acute myeloid

我们的实验室已经开始对p15INK4b进行系统分析，p15INK4b在人类急性髓性白血病（AML）中比任何其他具有细胞周期调节特性的肿瘤抑制基因更频繁地失活。p15Ink4b是一种周期蛋白依赖性激酶抑制剂，与其他Ink4家族成员一样，与早期G1周期蛋白依赖性激酶4和6结合，从而阻止它们与D周期蛋白的相互作用。我们先前提供的证据表明，该基因是小鼠髓性白血病的肿瘤抑制因子。首先，发现逆转录病毒诱导的髓细胞白血病的骨髓单核细胞表型在该基因的启动子区域具有高甲基化。在某些情况下，白血病在原发状态下没有高甲基化，在同基因小鼠移植后，肿瘤细胞发生甲基化。在p15ink4b缺陷小鼠中进行的第二种类型的研究提供了关于Ink4b肿瘤抑制作用的最令人信服的数据。在逆转录病毒诱导的白血病发生方案中使用小鼠表明，Ink4b缺陷杂合的小鼠对髓性白血病的易感性增加。小鼠接种了MOL4070LTR逆转录病毒，这是我们实验室最近构建和生产的一种独特的髓系疾病诱导病毒。逆转录病毒通过插入诱变提供了合作事件，使白血病的易感性证明了一个等位基因的丢失。缺乏两个等位基因的小鼠并不比缺乏一个等位基因的小鼠更容易受到影响，这增加了Ink4b缺失小鼠发生髓性白血病的可能性。为了帮助我们理解为什么Ink4b-/-小鼠并不比Ink4b-/+小鼠更容易患白血病，我们基于loxP-Cre系统开发了Ink4b的条件敲除。条件等位基因纯合的小鼠已经产生，并显示在正常水平上表达p15Ink4b。这些小鼠现在正在与Mx1Cre小鼠杂交，这将允许在用pI-pC处理小鼠后删除Ink4b的外显子2。此外，将具有条件敲除等位基因的小鼠与LysMCre小鼠杂交，在髓系谱系中进行特异性缺失。随后将分析小鼠对逆转录病毒诱导的白血病的易感性。在白血病中，造血干细胞和祖细胞之间增殖和分化的正常平衡可能被破坏。为了确定p15Ink4b是否在调节造血前体细胞水平中起作用，我们检测了Ink4b-/-小鼠的造血祖细胞池。在髓系祖细胞集落测定中，Ink4b-/-小鼠的骨髓中含有1.4倍多的致力于形成粒细胞和巨噬细胞（CFU-GM）的祖细胞。这一体外数据得到了流式细胞分析的支持，该分析确定Ink4b-/- BM含有3倍的粒细胞-巨噬细胞祖细胞（GMP）比例（11+/-1.2% vs 34+/-9%），同时普通髓系祖细胞（CMP）减少2倍（56+/- 2% vs 34+/- 7%）。从Ink4b-/-骨髓中分离的GMP也显示出3倍的形成CFU-GM的倾向。尽管存在这些差异，但通过碘化丙啶和hoecsht染色DNA染色以及体内BrdU掺入测定，Ink4b-/-和wt小鼠中循环GMP或CMP的比例是相同的。然而，在对特定细胞因子的反应中，Ink4b-/- CMP显示出GMP分化的潜力增加，而形成巨核红细胞祖细胞（MEP）的能力下降。我们的研究表明，p15Ink4b在维持正常水平的CFU-GM中起作用，并可能根据特定因素调节CMP分化。在另一个研究领域，我们的实验室检测了人类AML亚型INK4B位点的超甲基化。

Our laboratory has embarked on a systematic analysis of p15INK4b, which in human acute myeloid leukemia (AML) is inactivated more frequently than any other tumor suppressor gene with cell cycle regulatory properties. p15Ink4b is a cyclin-dependent kinase inhibitor and like other Ink4 family members binds to early G1 cyclin-dependent kinases 4 and 6, thereby preventing their interaction with D cyclins. We have previously provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice. First, retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation within the gene's promoter region. In some cases where leukemias did not have hypermethylation in the primary state, the neoplastic cells developed methylation following transplantation in syngeneic mice. A second type of study carried out in p15Ink4b-deficient mice provided the most convincing data regarding the tumor suppressor role of Ink4b. Use of the mice in a retrovirus-induced leukemogenesis protocol showed that mice heterozygous for Ink4b deficiency had increased susceptibility to myeloid leukemia. Mice were inoculated with retrovirus MOL4070LTR, a unique myeloid disease-inducing virus, which was recently constructed and produced in our laboratory. The retrovirus provided the cooperating events through insertional mutagenesis that allowed the demonstration of the susceptibility to leukemia provided by loss of one allele. Mice deficient in both alleles were not more susceptible than those deficient in one allele, raising the possibility that there are opposing forces to the development of myeloid leukemia in Ink4b null mice. To assist in our understanding of why Ink4b-/- mice are not more susceptible to leukemia than Ink4b-/+ mice we have developed a conditional knockout of Ink4b based on the loxP-Cre system. Mice, homozygous for the conditional allele, have been generated and shown to express p15Ink4b at normal levels. These mice are now being crossed with Mx1Cre mice which will allow deletion of exon 2 of Ink4b following treatment of mice with pI-pC. In addition, mice with the conditional knockout allele are being crossed with LysMCre mice for specific deletion in the myeloid lineage. Mice will be subsequently analyzed for susceptibility to retrovirus-induced leukemia.In leukemia, the normal balance between the proliferation and differentiation of hematopoietic stem and progenitor cells can be disrupted. To determine if p15Ink4b plays a role in regulating the levels hematopoietic precursors, we examined the hematopoietic progenitor pool in Ink4b-/- mice. In myeloid progenitor colony assays, bone marrow from Ink4b-/- mice were found to contain a 1.4 fold greater number of progenitors committed to the formation of granulocytes and macrophages (CFU-GM). This in vitro data was supported by flow cytometric analysis which determined that Ink4b-/- BM contained a 3-fold greater proportion of granulocyte-macrophage progenitors (GMP) (11+/-1.2% vs 34+/-9%) concomitant with a 2-fold decrease in common myeloid progenitors (CMP) (56+/-2 % vs 34+/-7 %). GMP isolated from Ink4b-/- bone marrow also demonstrated a 3-fold greater propensity to form CFU-GM. Despite these differences, the proportion of cycling GMP or CMP in Ink4b-/- and wt mice were identical as determined by propidium iodide and hoecsht dye DNA stains as well as in vivo BrdU incorporation. However, in response to specific cytokines, Ink4b-/- CMP showed an increased potential for GMP differentiation and a decreased capacity to form megakaryocyte-erythroid progenitors (MEP). Our work demonstrates that p15Ink4b functions in maintaining normal levels of CFU-GM and may regulate CMP differentiation in response to specific factors. In another area of research our laboratory has examined human AML subtypes for hypermethylation of the INK4B locus.