GENES CONTROLLING NEOPLASIA OF THE INTESTINAL EPITHELIUM

控制肠上皮肿瘤的基因

• 批准号: 7175323
• 负责人: WILLIAM Franklin DOVE
• 金额: $ 87.81万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-22 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7175323
• 关键词: Affect; Aggressive Fibromatosis; Alleles; Animals; Apoptotic; Biological Markers; Cell Cycle; Chromosomal Instability; Chromosomes, Human, Pair 18; Clonality; Colonic Adenoma; Condition; Cytosine; DNA; DNA Modification Methylases; DNA Sequence; Defect; Disease regression; Enhancers; Epigenetic Process; Gene Silencing; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genome; Genotype; Goals; Growth; Heterozygote; Human; Image; Intestinal Neoplasms; Intestines; Invasive; Knock-out; Life; Link; Localized; Longitudinal Studies; Loss of Heterozygosity; Maintenance; Maps; Mass Spectrum Analysis; Methods; Methylation; Methyltransferase; Mismatch Repair; Mitotic; Molecular; Mouse Strains; Mus; Mutagenesis; Mutagenicity Tests; Mutagens; Mutation; Paneth Cells; Pathway interactions; Phenotype; Phospholipase; Play; Positioning Attribute; Protein p53; Rate; Resistance; Resources; Retinoblastoma; Role; Serum; Site-Specific DNA Methyltransferase (Cytosine-Specific); Staging; Stem cells; TP53 gene; Testing; Transforming Growth Factor alpha; Tumor Suppressor Genes; Work; adenoma; base; chromosome loss; fibroma; intestinal epithelium; member; mutant; promoter; segregation; tumor; tumor growth

Over the past several years, we have developed a unique ensemble of resources by which to explore fundamental issues in the earliest stage of intestinal neoplasia, the formation and maintenance of the adenoma. Our studies in the Min mouse strain have highlighted two new pathways by which the activity of the tumor suppressor gene Apc is lost: homologous somatic recombination and apparent epigenetic silencing. These early pathways do not involve the canonical genetic instability mechanisms involving mismatch repair or chromosome loss. We are now positioned to search for polymorphisms in the mouse and human that affect somatic recombination or epigenetic silencing. These studies have also highlighted the preponderance of polyclonality in the early intestinal adenoma. We now aim to investigate whether this polyclonality extends to tumors that involve epigenetic silencing and desmoid fibromas associated with defects in the Apc and p53 tumor suppressor genes. Finally, our studies of the familial intestinal adenoma in the mouse have identified two molecular modifiers of net tumor growth rate - DNA cytosine methylase and a secretory phospholipase. Though each modifier has only modest activity when introduced singly, in combination they show very strong synergy. We shall investigate the molecular basis of such synergy and explore how broadly it is found. We have positioned ourselves to pursue the more global identification of molecular modifiers through mutagenesis of the mouse germline and the use of an isogenic strategy that avoids the complications of abundant polymorphic modifiers. Intestinal neoplasia becomes life-threatening when it progresses to invasive and metastatic forms. We have established conditions in which the tumors of Min mice are locally invasive and methods for MicroCT imaging that permit longitudinal studies of progression and regression. Over the coming years, we aim to deepen our studies of early and late intestinal neoplasms by seeking molecular markers expressed within tumors and in the serum of tumor-bearing animals. The former goal will be explored not only by array analysis, but also by retroviral tagging. The latter goal will be explored by the emergent power of mass spectrometry. AIMS Aim la: Does a total deletion of Apc show the classical Min phenotype in heterozygotes, Apcde'/+? Aim 1 b: In ApcMin/+ (Mid+) heterozygotes do any adenomas form that retain an active, functional wildtype Apc allele? Aim Ic Does desmoid fibroma formation in Mid+ heterozygotes also involve loss of the wildtype Apc allele? Aim Id: Does the silencing pathway involve methylation of the promoter controlling expression of the wildtype Apc allele? Aim le: Is silencing limited to the wildtype allele, or does it also affect the Min mutant allele of Apc? Aim If: Is there a heterozygous effect of the Min nonsense allele that predisposes to adenoma formation? Aim 2a: Is early adenoma formation in the Min/+ intestinal epithelium polyclonal (cf Novelli et al., 1996)? What is the clonality of the contrasting Min-induced desmoid fibroma when localized and when invasive? Aim 2b: Are rates of mitotic segregation affected by the Apc genotype or by the genetic background? Aim 2c: Does transforming growth factor alpha (TGFa) affect the multiplicity, phenotype and clonality of Min-induced adenomas? Aim 2d: In the Min mouse, does homologous somatic recombination play a major role in loss-of-heterozygosity, as opposed to chromosomal instability? Aim 2e: Are the allele losses that are seen in human colonic adenomas also a result of homologous somatic recombination? Aim 3a: Is the secretory phospholipase (PlaZgZa; see MacPhee et al., 1995, and Gould et al., 199613) a component of the modifier-of-Min, Mom?? Do the components of Mom7 (Mom7A and Mom?@ correspond to members of the cluster of phospholipases in this region of the genome? Aim 3b: While working toward the analysis of Mom? at the molecular level, to establish which allele of Mom7A and of Mom7B is functional, resistance or sensitivity? Aim 3c: Is the Min phenotype modified in animals homozygous for a targeted allele that inactivates Wnt2 and/or in animals heterozygous for a P-catenin mutation? Aim 3d: Do any of the known molecular modifiers of the Min phenotype Morn? affect the proliferative and apoptotic dynamics of the stem cell and proliferative region of the normal intestinal epithelium or within the nascent adenoma? Aim 3e: Does the action of any of these modifiers depend upon the Paneth cell compartment? Aim 4a: Does the Mom? effect on the adenoma pathway depend upon wildtype p53 function? Does Mom1 affect desmoid tumor formation or growth? Aim 4b: Does DNA cytosine methyltransferase (Dnmt) deficiency affect the desmoid pathway? Aim 4c: Do Morn? and Dnmt act independently on the adenoma pathway? Aim 5a: To validate and characterize further a set of candidate dominant Enhancers (5) and Suppressors (7) of the Min survival phenotype found in our initial sensitized two-generation screen for mutagen-induced modifier alleles. Aim 5b: To develop the screen in a compact form to facilitate the isolation and mapping of mutagen-induced dominant Enhancers and Suppressors lying in vital genes. Aim 5c: For a DNA sequence whose methylation status decreases in Min induced adenomas, test whether targeted inactivation of the gene enhances the Min phenotype. retinoblastoma cell cycle regulator Rb influences the Min phenotype. Aim 5d: Investigate whether heterozygosity for a knockout allele of the retinoblastoma cell cycle regulator Rb influences the Min phenotype. Aim 5e: Analyze two new polymorphic modifier loci linked to the Apc locus on mouse chromosome 18 - a recessive enhancing allele in the strain BTBR, and a semidominant resistance allele in the strain DBN2.

在过去的几年中，我们开发了一种独特的资源集合，可以在肠道肿瘤的最早阶段探索基本问题，腺瘤的形成和维护。我们在最小小鼠菌株中的研究强调了两种新的途径，肿瘤抑制基因APC的活性丢失了：同源的体细胞重组和明显的表观遗传沉默。这些早期途径不涉及涉及不匹配修复或的规范遗传不稳定性机制 染色体损失。现在，我们可以搜索影响体细胞重组或表观遗传沉默的小鼠和人类中的多态性。这些研究还强调了早期肠道腺瘤中多克隆性的优势。现在，我们旨在研究这种多克隆性是否扩展到涉及表观遗传沉默和 与APC和p53肿瘤抑制基因缺陷相关的脱粒纤维瘤。最后，我们对小鼠家族性肠道腺瘤的研究已经确定了两个分子修饰剂 净肿瘤生长速率 - DNA胞嘧啶甲基酶和分泌磷脂酶。尽管每个修饰符单都只有单独引入时才适度的活动，但结合起来，它们表现出非常强大的协同作用。我们将研究这种协同作用的分子基础，并探索发现它的范围。我们已经定位 通过小鼠种系的诱变以及使用的ISEOGONIC策略来追求对分子修饰剂的更全球鉴定，从而避免了丰富的多态性修饰剂的并发症。当肠道肿瘤发展为侵入性和转移形式时，它会威胁生命。我们有 建立的条件是，最小小鼠的肿瘤是局部侵入性的，以及用于允许对进展和消退的纵向研究的微分离成像的方法。在接下来的几年中，我们旨在通过寻求在肿瘤中和肿瘤动物的血清中表达的分子标记来加深对早期和晚期肠道肿瘤的研究。前者的目标不仅将通过阵列分析来探索，还可以通过逆转录病毒标记来探索。质谱的新兴力量将探索后一个目标。 目标 AIM LA：APC的总删除是否显示了杂合子中的经典最小表型，APCDE'/+？ AIM 1 B：在APCMIN/+（MID+）杂合子中，是否会保留活跃的功能性野生型APC等位基因的任何腺瘤形式？ AIM IC在MID+杂合子中的脱粒纤维瘤形成是否涉及野生型APC等位基因的丧失？ AIM ID：沉默途径是否涉及控制野生型APC等位基因表达的启动子的甲基化？ AIM LE：沉默是否仅限于Wildtype等位基因，还是会影响APC的最小突变等位基因？ 目的如果：min废话等位基因是否存在杂合作用 易感腺瘤形成？ AIM 2A：最小/+肠上皮多克隆中的早期腺瘤是否形成（CF Novelli等，1996）？对比鲜明的最小诱导的脱粒纤维瘤的克隆性是什么？ AIM 2B：是受APC基因型影响还是受遗传背景影响的有丝分裂偏析速率？ AIM 2C：转化生长因子α（TGFA）是否会影响最小诱导的腺瘤的多样性，表型和克隆性？ AIM 2D：在Min Mouse中，同源的体细胞重组在杂合性丧失中是否起着重要作用，而不是染色体不稳定性？ AIM 2E：在人类结肠腺瘤中看到的等位基因损失也是同源体细胞重组的结果吗？ AIM 3A：分泌磷脂酶（Plazgza;参见Macphee等，1995，and Gould等，199613）是米尔的修饰符，妈妈的组成部分？ MOM7的成分（MOM7A和MOM？@@对应于基因组该区域中磷脂酶簇的成员吗？ AIM 3B：在努力分析妈妈的同时？在分子水平上，为了确定MOM7A和MOM7B的哪个等位基因具有功能，抗性或灵敏度？ AIM 3C：在动物中，Min表型是否在纯合等等位基因中改变了靶标等位基因，该等位基因使Wnt2和/或动物杂合子中的动物杂合子突变？ AIM 3D：Min表型早晨的任何已知分子修饰剂中有什么？会影响正常肠上皮的干细胞的增殖和凋亡动力学，或者在开始腺瘤中的增殖区域吗？ AIM 3E：这些修饰符中的任何一个的作用是否取决于Paneth Cell室？ AIM 4A：妈妈吗？对腺瘤途径的影响取决于野生型p53功能？ MOM1会影响脱蛋白肿瘤形成还是生长？ AIM 4B：DNA胞嘧啶甲基转移酶（DNMT）缺乏会影响脱粒途径吗？ AIM 4C：早上吗？ DNMT在腺瘤途径上独立起作用？ AIM 5A：验证和表征进一步的一组候选增强子（5）（5）和抑制剂（7）（7）在我们在我们的初始敏化的两生屏幕上发现的诱变的修饰剂等位基因。 AIM 5B：以紧凑的形式开发屏幕，以促进诱发诱发的主要增强子和位于重要基因的抑制器的隔离和映射。 AIM 5C：对于一个DNA序列，其甲基化状态在最小诱导的腺瘤中降低的DNA序列，测试基因的靶向失活是否会增强最小表型。视网膜细胞瘤细胞周期调节剂RB影响最小表型。 AIM 5D：研究视网膜母细胞瘤细胞周期调节剂RB的敲除等位基因的杂合性是否会影响最小表型。 AIM 5E：分析两个新的多态性修饰剂基因座与小鼠染色体上的APC基因座相关联 - 菌株BTBR中的隐性增强等位基因，以及菌株DBN2中的半主导抗性等位基因。