Molecular Cytogenetics of Solid Tumors

尽管去年我们的项目在实验室的实地考察中得到了评估，但我们成功地获得了大量与肿瘤发展初始阶段相关的新数据，并具有用于治疗干预的潜力。癌症特异性易位、癌基因扩增、肿瘤抑制基因缺失和脆弱位点（FSs）病毒整合的分子和细胞遗传学证据构成了将脆弱和重组基因组区域与癌症发展联系起来的转折点。很早以前，我们就提出了将FS作为转化病毒整合的优先靶点的概念。这一观点目前已被普遍接受。本文提供了一个新的例子，证明了病毒在FS上的特异性整合作用，显示了猴病毒40 （SV 40）在位于染色体1q21上的一个FRA1F上的整合。SV40在细胞基因组中的插入导致了与衰老和凋亡有关的基因的失调，因此在细胞永生中起着关键作用。随后，分离出SV40基因组侧翼的人类基因组序列，并鉴定出150 KB的BAC，与1q21的病毒整合位点相对应。用neo标记对BAC克隆进行改良，并将其引入亲本不朽细胞系。BAC转移克隆表现出正常成纤维细胞的生长特征，以复制性衰老终止。这些结果表明，被病毒插入破坏的基因的异位表达可以恢复正常的生长模式和衰老。围绕BAC克隆的1q21区域的高分辨率图谱确定了在许多癌症中重排或显示异常表达的基因，并提示该区域在癌症发展中的作用。在过去的几年中，我们在胚胎干细胞的细胞遗传学方面获得了相当多的经验。这种专门知识引起了几位校内外科学家的兴趣。其中一项成果是与来自巴尔的摩NIDA分子神经生物学分支的Uhls博士小组合作，培育出了KEPI基因敲除小鼠。KEPI是一种吗啡调节基因，是一种有效的蛋白磷酸酶1抑制剂。该基因定位到小鼠10号染色体上，靠近含有-阿片受体（Oprm1）的位点。最终构建完成后，将pJD9与Not I线性化，电孔插入129.3个小鼠MC1胚胎干（ES）中，利用引物vNEOf和J39对376个抗G418菌落进行PCR筛选。PCR阳性的胚胎干细胞克隆经Southern blot进一步证实。8个正确靶向的ES细胞克隆进行了广泛的核型分析，其中2个证实没有非整倍体和结构缺陷，将其微注射到C57BL/6J E3.5囊胚中。将得到的KEPI KO杂合雄性嵌合体进行杂交，同时扩增野生型（WT）和KO （922 bp）等位基因。在过去的十年中，人类DLC-1（在肝癌中删除1）基因已成为一种有效的肿瘤抑制基因，也是癌症中最常被解除调控的基因之一，在与多种癌症相关的前50个基因中排名第五。dcl1功能丧失被认为是促进和进展肝脏和其他癌症的驱动事件。鉴定并鉴定了一种新的dcl1亚型4 （DLC1-i4）。与其他亚型相比，DLC1-i4编码一个具有不同n端的1125-aa蛋白，并且与其他亚型一样，在正常组织和永生化正常上皮细胞中普遍表达，这表明它是DLC1的主要转录物。然而，在肿瘤细胞系中发现了四种dcl1亚型的差异表达：亚型1和亚型3（可能无功能）共享一个启动子，并且在几乎所有癌症和永生化细胞系中沉默，而亚型2和亚型4使用不同的启动子并且经常下调。DLC1-i4在大量鼻咽癌、食管癌、胃癌、乳腺癌、结直肠癌、宫颈癌和肺癌细胞系以及原发肿瘤中均显著下调。功能性DLC1-i4启动子位于CpG岛内，由野生型p53激活。5-aza-2-脱氧胞苷处理导致启动子的去甲基化和DLC1-i4表达的再激活。异位表达isofor4在dlc1 -i4阴性肿瘤细胞中强烈抑制其生长和集落形成，表明该异构体具有抑癌作用。各种DLC1亚型的差异表达表明，在致癌过程中，DLC1的复杂活性在调节中相互作用。肿瘤抑制基因的失活是癌症发生或发展的一个重要改变。与大多数其他肿瘤抑制基因相比，dcl1在各种常见癌症中更频繁地缺失，然而dcl1的下调和失活主要是由启动子超甲基化和组蛋白去乙酰化介导的。由于DNA甲基转移酶和组蛋白去乙酰化酶（HDAC）抑制剂可以诱导DLC-1表达的恢复，DLC-1蛋白也可能是新疗法的潜在靶点。鉴于表观遗传治疗的巨大兴趣和进展，许多有前途的抗肿瘤药物，特别是HDAC抑制剂，已经开发并成功用于临床试验。DLC1和HDAC抑制剂都具有抗肿瘤功能，它们的联合作用可以用于更有效的癌症治疗。为了评估这种方法的潜在益处，我们检测了腺病毒(Ad)- dlc1介导的转导的抗肿瘤作用，并在两种dlc1阴性的人类癌细胞系- 22Rv1（前列腺癌）和7703K（人肝细胞癌）细胞中暴露于亚eroylanilide - hydroxyamic acid (SAHA)，一种强大的HDAC抑制剂。与dcl1在多种癌症中的抑癌功能一致，用腺病毒ad - dcl1表达载体转导前列腺和肝癌细胞可导致细胞形态改变，诱导凋亡，抑制细胞增殖、迁移和非锚定生长。低浓度的SAHA可以有效地恢复由于组蛋白去乙酰化而缺乏DLC1表达的前列腺肿瘤细胞中DLC1的表达，但对主要由于启动子超甲基化而导致DLC1基因沉默的肝肿瘤细胞影响甚微。无论DLC1失活的表观遗传机制如何，SAHA处理DLC1转导细胞对两种细胞系的肿瘤细胞增殖和肿瘤发生均有协同抑制作用。在前列腺肿瘤细胞中，这种联合治疗方案实际上消除了半固体培养基中菌落的形成，作为体外致瘤性的衡量标准。目前的体外实验结果验证了该方案作为某些癌症的潜在新治疗选择。

Although last year our program was evaluated at the Site Visit of the laboratory, we succeeded to generate significant pool of new data withy relevance to initial stages of neoplastic development and with potential for use in therapeutic interventions. The molecular and cytogenetic evidence for cancer-specific translocations, amplification of oncogenes, deletion of tumor suppressor gene and viral integration at fragile sites (FSs) constitute the turning point in linking genomic regions of fragility and recombination to cancer development. Long time ago, we advanced the concept of FS as preferential target for integration of transforming viruses. This notion is currently generally accepted. A novel example of the role of specific viral integration at FS was provided showing Simian virus 40 (SV 40) integration at a FRA1F located on chromosome 1q21. SV40 insertion in the cells genome led to deregulation of genes involved in senescence and apotosis and thus plays a critical role in cellular immortalization. Subsequently a human genomic sequences flanking SV40 genome was isolated and identified a 150 KB BAC, corresponding to the viral integration site at 1q21. The BAC clone was retrofitted with a neo marker for selection and introduced into the parental immortal cell line. BAC transfer clones displayed growth characteristics of normal fibroblasts terminating in replicative senescence. These results show that ectopic expression of gene(s) disrupted by viral insertion restore normal growth pattern and senescence. A high-resolution map of 1q21 region, surrounding the BAC clone, identified genes that are either rearranged or show abnormal expression in many cancers and suggests a role for this region in the development of cancer. In the past few years we gained considerable experience in cytogenetics of embryonic stem cells. This expertise attracted the interest of several intra- and extramural scientists. One of the results is the generation of a KEPI knockout mouse in a collaboration with Dr. Uhls group from the Molecular Neurobiology Branch, NIDA, in Baltimore. KEPI as a morphine-regulated gene, which is a powerful inhibitor of protein phosphatase 1. The gene maps onto mouse chromosome 10 close to the locus that contains the -opioid receptor (Oprm1). After the final construct, pJD9 was linearized with Not I and electroporate into 129.3 mouse MC1 embryonic stem (ES), cells, primers vNEOf and J39 were used for a PCR screen of 376 colonies resistant to G418. Embryonic stem (ES) cell clones positive by PCR were further confirmed by Southern blot. Eight of the correctly targeted ES cell clones were extensively karyotyped and the two confirmed free of aneuploidy and structural deffects were microinjected into C57BL/6J E3.5 blastocysts. Heterozygous KEPI KO offspring of the resulting male chimeras were mated with each other, to amplify wild type (WT) and KO, (922 bp) alleles simultaneously. Recombinant, KEPIKO mice displayed set of characteristsics that support roles for KEPI gene action in adaptive responses Over past decade the human DLC-1 (Deleted in Liver Cancer 1) gene has emerged as an potent tumor suppressor gene and as one of the most frequently deregulated genes in cancer, being the 5th among top-50 genes implicated in multiple cancers. DLC1 loss of function is viewed as a driving event in the promotion and progression of liver and other cancers. A novel DLC1 isoform 4 (DLC1-i4) was identified and characterized. The DLC1-i4 encodes an 1125-aa protein with distinct N-terminus compared to the other isoforms and, as others, is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. However, differential expression of the four DLC1 isoforms is found in tumor cell lines: Isoform 1 and 3 (probably nonfunctional) share a promoter and are silenced in almost all cancer and immortalized cell lines, while isoform 2 and 4 utilize different promoters and are frequently downregulated. DLC1-i4 is significantly down-regulated in a high number of nasopharyngeal, esophageal, gastric, breast, colorectal, cervical and lung carcinoma cell lines as well as in primary tumors. The functional DLC1-i4 promoter is within a CpG island and is activated by wild-type p53. Treatment with 5-aza-2-deoxycytidine led to demethylation of the promoter and reactivation of DLC1-i4 expression. Ectopic expression of isofor 4 in DLC1-i4-negative tumor cells strongly inhibited their growth and colony formation showing that this isoform has oncosuppressive role. The differential expression of various DLC1 isoforms suggests interplay in modulating the complex activities of DLC1 during carcinogenesis. Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. DLC1 is deleted in various common cancers more frequently than most other tumor suppressors genes, however down regulation and inactivation of DLC1 is mediated predominantly by promoter hypermethylation and histone deacetylation. Because DNA methyltransferase and histone deacetylase (HDAC) inhibitors can induce the restoration of DLC-1 expression, the DLC-1 protein may also represent a potential target for novel therapies. Given the considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly HDAC inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction, and the exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor in two DLC1-negative human cancer cell lines - 22Rv1 (prostate cancer) and 7703K (human hepatocellular carcinoma) cells. Consistent with the oncosuppressive function of DLC1 in several cancers, transduction of prostate and liver cancer cells with an adenovirus AD-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA efficiently restored the expression of DLC1 in prostate tumor cells that lack DLC1 expression due to histone deacetylation, but had a minimal effect in liver tumor cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In prostate tumor cells, this combination regimen virtually abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.