Oncogenes and tumor suppressors in murine acute myeloid

最近，我们一直在关注肿瘤抑制因子在我们实验室建立的髓系白血病模型中的作用。细胞周期蛋白依赖性激酶(CDK)抑制剂是一种在细胞周期控制中发挥重要作用的肿瘤抑制物，在人类白血病中已通过缺失、启动子超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。 在一个研究领域，我们已经完成了对c-Myb和c-Myc诱导的髓系白血病INK4基因变化的检查。内源性c-myb通过逆转录病毒插入突变诱导的肿瘤一般不表达p15INK4b 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。与Myb肿瘤不同，携带c-myc基因的逆转录病毒诱导的Myc肿瘤一般表达p15INK4b RNA和蛋白，提示c-Myc可以绕过这些抑癌基因的作用。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？P19Arf。有趣的是，大多数Myc肿瘤都缺失了p19Arf外显子，甚至在某些情况下p16INK4a没有缺失。因此，我们的数据表明，在Myc诱导的肿瘤中，CDKN4a基因座的变化可能主要是由于细胞试图失活p19ARF，从而使P53肿瘤抑制通路失活。 该实验室现已开始对p15INK4b进行系统分析。我们最近创造了一种新的重组逆转录病毒，名为MOL4070LTR，其独特之处在于它能够感染所有品系的小鼠，并以大致相等的比例诱发髓系和淋巴系疾病。感染这种病毒被证明是非常有用的诱导髓系白血病的模型，通过p15INK4b基因的高甲基化诱导白血病，从而模拟人类的情况。该实验室正在确定甲基化的共同起始位置，并分析甲基化在这些白血病中通过基因扩展启动子区的传播。此外，我们正在将特定的甲基化模式与从原发肿瘤阶段进展到移植和组织培养建立过程中的转录联系起来。为了在小鼠中展示p15INK4b的肿瘤抑制功能，我们开发了一种靶向缺失该基因的小鼠。得到的小鼠已经与MOL4070LTR白血病逆转录病毒的接种结合使用。初步实验正在提供初步数据，表明p15INK4b是髓系白血病发生的抑制因子，因为具有靶向缺失的小鼠的白血病发病率高于野生型小鼠。

Recently we have been focusing on the role of tumor suppressors in models of myeloid leukemia developed in our laboratory. Cyclin dependent kinase (cdk) inhibitors are tumor suppresses that play an important role in cell cycle control and have been inactivated in human leukemias 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 we have completed an examination of myeloid leukemias induced by c-Myb and c-Myc for alterations in INK4 gene. The neoplasms induced by endogenous c-myb, through retroviral insertional mutagenesis generally do not express p15Ink4b RNA. A lack of expression in the Myb tumors is not due to deletion or methylation. Further studies in vitro have indicate that the c-Myb itself can repress the expression of p15INK4b. The Myc tumors that were induced by a retrovirus carrying the c-myc gene, in contrast to the Myb tumors general do express p15Ink4b RNA and protein, suggesting that c-Myc can bypass the effects of these tumor suppressor. 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. Interestingly, most of the Myc tumors have deleted p19Arf exons even in some cases where p16Ink4a is not deleted. Therefore, our data would suggest that alterations in the Cdkn4a locus in the Myc induced tumors may primarily be due to attempts by the cell to inactivate p19ARF and, therefore, the p53 tumor suppressor pathway. The laboratory has now embarked on a systematic analysis of p15Ink4b. We recently created a new recombinant retrovirus, called MOL4070LTR, that is unique in its ability to infect all strains of mice and to induce myeloid and lymphoid disease in approximately equal proportions. Infection with this virus, which is proving to be extremely useful as a model for induction of myeloid leukemias, induces leukemias with hypermethylation of the p15Ink4b gene, thus mimicking the situation in man. The lab is in the process of determining the common initiation sites for methylation and analyzing the spread of methylation through the extended promoter region of the gene in these leukemias. In addition, we are correlating specific methylation patterns with transcription during progression from the primary tumor stage to transplantation and tissue culture establishment. For the purpose of demonstrating a tumor suppressor function for p15Ink4b in mice, we have developed a mouse with targeted deletion of this gene. The resulting mouse has been used in combination with inoculation of the MOL4070LTR leukemogenic retrovirus. Initial experiments are providing the preliminary data to suggest that p15Ink4b is a suppressor of myeloid leukemogenesis, because the incidence of leukemia is higher in mice with the targeted deletion than it is in wild-type mice.