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Characterizations of chromosome aberrations, telomere dysfunction, and radiosensitivity signatures in canine cancer cell lines.

机译:犬癌细胞系中染色体畸变,端粒功能障碍和放射敏感性特征的表征。

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摘要

Cancer is now a leading cause of death in dogs as well as in humans due to longer life spans resulting from advances in nutrition and veterinary medicine, and the higher expectations of pet owners. Companion dogs share environmental influences with humans and spontaneously develop tumors with histopathologic and biologic behavior similar to tumors that occur in humans. Therefore, canine cancer research has much potential to benefit both dogs and humans.;Cancer cell lines have been widely used as in vitro experimental model systems and have proven to be useful for exploring the underlying biology of cancer. Canine cancer cell lines have increasingly been developed and utilized, but are not as fully characterized as human cell lines. In this thesis, we characterized canine cancer cell lines by examining: 1) chromosome aberrations, long appreciated as valuable biomarkers of carcinogenesis; 2) telomeres, chromosomal features with important implications for both aging and cancer; and 3) cellular radiosensitivity signatures, critical for evaluating individual response to radiation therapy. Such characterization of canine cancer cell lines will provide a better understanding of underlying canine cancer biology and provide new insights into improved clinical management, such as development of novel therapeutic targets and identification of radiation sensitivity markers, not only for dogs, but of potential relevance for humans as well.;First, we investigated chromosome and telomere aberrations in canine osteosarcoma (OSA) cell lines, a common primary bone tumor in both humans and dogs. Previously, malignant canine cancer cells have been reported to exhibit metacentric chromosomes (morphologically irregular chromosomes for canines) in a range of canine tumors including osteosarcoma. The metacentric chromosomes, which likely represent end-to-end chromosomal fusion events (Robertsonian translocations), may reflect telomere dysfunction, leading us to hypothesize that chromosome aberrations involving uncapped telomeres may be a common feature of canine OSA cells. Eight established canine OSA cell lines were evaluated; chromosome number and frequency of metacentric chromosomes were determined in metaphase spreads. Using fluorescence in situ hybridization (FISH) with a telomere specific probe, the contribution of dysfunctional telomeres was characterized. We also assessed telomere associated factors including telomerase activity, co-localization of DNA damage and telomere signals (telomere-dysfunction induced foci, TIFs), and expression of a DNA repair protein shown to be required for mammalian telomeric end-capping function, specifically the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Despite variable chromosome numbers, proliferation rates and radiosensitivities, all eight canine OSA cell lines displayed increased numbers of metacentric chromosomes and exhibited numerous telomere aberrations including interstitial telomeric signals and telomere fusions (telomere signals at the point of fusion, in this case in the centromeric regions). D17, the oldest canine OSA cell line used in the study, showed the highest frequency of telomere aberrations (51.4 per cell) and metacentric chromosomes (43.2 per cell). Furthermore, the cell lines were all telomerase positive and showed no correlations between their telomere aberrations and the other telomere associated factors analyzed. To better characterize the telomere dysfunction associated with canine OSA, we investigated telomere aberrations in primary cultures from ten spontaneous canine OSAs, as well as the effects of long-term culture. Seven of the primary samples displayed no increase in frequency of metacentric chromosomes, while three of the samples did have elevated levels (11.5 per cell in the highest primary culture); however, no telomere signals were present at the involved centromeric regions. Telomere aberrations were observed in all of the primary cultures, but the number was small (1.87 per cell in the highest primary culture). Interestingly, we found that metacentric chromosome frequencies increased in one primary OSA culture with increasing passage in culture. In contrast, metacentric chromosomes did not accumulate with long-term culture of non-cancerous canine fibroblasts, or DNA repair deficient mouse fibroblasts (homozygous ataxia telangiectasia mutated (ATM) gene deficiency). Together, these results suggested that metacentric chromosomes and telomere dysfunction of are characteristics of canine OSA. Therefore, targeting of these unique telomere aberrations has potential for both improving diagnosis of canine OSA, as well as development of novel treatment strategies.;Lastly, we investigated radiosensitivity in a panel of canine cancer cell lines representing different cancer types. A human cancer cell line panel, NCI-60, consisting of different tumor types has been successfully utilized for in vitro anticancer drug screening, demonstrating the utility of such an approach in understanding cancer mechanisms; gene expression profiling and variable radiosensitivities facilitated development of informative markers regardless of tumor tissue type. A canine cancer cell line panel, called the ACC30, was recently developed at the Flint Animal Cancer Center (FACC), Colorado State University and includes microarray gene expression data. We aimed to test the hypothesis that identification of the determinants of response to ionizing radiation in the diverse canine cancer cell lines would provide additional information, some possibly specific to dogs, and some potentially supplementing those reported for human cancer. We obtained 27 canine cell lines derived from ten tumor types from the ACC30 panel. First, radiosensitivity was determined using a clonogenic assay for adherent cell cultures, or a limiting dilution assay for suspension cell cultures. The 27 cell lines had varying radiosensitivities regardless tumor type (survival fraction at 2 Gy, SF2= 0.2--0.9). Based on the cellular characteristics analyzed in this study, there was a moderate correlation between radioresistance and better plating efficiency in the 27 cell lines. Next, we selected the six most radiosensitive cell lines as the radiosensitive group and the five most radioresistant cell lines as the radioresistant group. Then, we evaluated known parameters for cell killing by ionizing radiation (IR) including IR-induced DNA double strand break (DSB) repair and apoptosis, in the radiosensitive group as compared to the radioresistant group. However, the two groups were not distinguished by these parameters. Further, we investigated a possible common radiosensitivity signature using the basal gene expression profiling of the ACC panel for 20,000 genes. More than 550 genes were identified as being differentially expressed between the radiosensitive and radioresistant groups. Gene set enrichment analysis was used to inform potential pathways and functions involving the differentially expressed genes and indeed, several biological processes including cell adhesion, cell migration and apoptosis were related to radiosensitivity in the canine cancer cell lines. In support of our findings, cell adhesion was one of the signatures previously identified in the human microarray analysis. Together, our results suggest that cell adhesion related genes, rather than the more commonly regarded radiosensitivity associated apoptosis and DNA repair related genes, may provide beneficial radiosensitivity biomarkers for predicting individual response to radiotherapy, regardless of tumor type. Thus, the radiosensitivity signatures characterized here may help guide future development of intrinsic tumor radiosensitivity biomarkers for predictive assays in canine cancer, and has the potential to improve predicting radiosensitivity in human cancer as well.
机译:由于营养和兽医学的进步以及宠物主人的更高期望而导致更长的寿命,癌症已成为狗和人类死亡的主要原因。伴侣犬与人类共享环境影响,并自发发展出具有类似于人类肿瘤的组织病理学和生物学行为的肿瘤。因此,犬类癌症的研究对狗和人类都有很大的益处。癌细胞系已被广泛用作体外实验模型系统,并被证明对探索癌症的基本生物学有用。犬癌细胞系已被越来越多地开发和利用,但不像人类细胞系那样被充分表征。在本文中,我们通过检查以下特征来鉴定犬癌细胞系:1)染色体畸变,长期以来被认为是有价值的致癌生物​​标志物; 2)端粒,染色体特征对衰老和癌症都有重要影响; 3)细胞放射敏感性特征,对于评估个体对放射治疗的反应至关重要。犬癌细胞系的这种表征将提供对犬癌基础生物学的更好理解,并为改进临床管理提供新见解,例如开发新的治疗靶标和鉴定放射敏感性标记物,不仅适用于狗,而且与犬的潜在相关性首先,我们研究了犬骨肉瘤(OSA)细胞系(人和狗中常见的原发性骨肿瘤)中的染色体和端粒畸变。以前,已经报道恶性犬癌细胞在包括骨肉瘤在内的一系列犬肿瘤中表现出亚中心染色体(犬的形态上不规则的染色体)。亚中心染色体很可能代表端到端的染色体融合事件(罗伯逊易位),可能反映端粒功能障碍,使我们推测涉及未封闭端粒的染色体畸变可能是犬OSA细胞的常见特征。评价了八种建立的犬OSA细胞系;在中期扩散中确定了亚中心染色体的染色体数目和频率。使用端粒特异性探针的荧光原位杂交(FISH),对功能异常的端粒的贡献进行了表征。我们还评估了端粒相关因素,包括端粒酶活性,DNA损伤和端粒信号的共定位(端粒功能障碍引起的病灶,TIF),以及哺乳动物端粒封端功能所必需的DNA修复蛋白的表达,特别是DNA依赖性蛋白激酶催化亚基(DNA-PKcs)。尽管染色体数目,增殖率和放射敏感性不同,但所有八种犬OSA细胞系均显示出偏中心染色体数目增加,并表现出许多端粒畸变,包括间质端粒信号和端粒融合(融合点处的端粒信号,在这种情况下为着丝粒区域) )。 D17是研究中使用的最古老的犬OSA细胞系,显示出端粒畸变(每个细胞51.4个)和偏中心染色体(每个细胞43.2个)的出现频率最高。此外,细胞系均为端粒酶阳性,并且其端粒畸变与所分析的其他端粒相关因子之间无相关性。为了更好地表征与犬OSA相关的端粒功能障碍,我们研究了来自十种自发犬OSA的原代培养中的端粒畸变,以及长期培养的影响。七个原始样本显示亚中心染色体的频率没有增加,而三个样本确实有升高的水平(在最高的原始培养物中每个细胞为11.5)。然而,在所涉及的着丝粒区域没有端粒信号。在所有原代培养物中均观察到端粒畸变,但数量很小(在最高原代培养物中每细胞1.87)。有趣的是,我们发现在一种主要的OSA培养物中,亚中心染色体的频率随着培养物中传代的增加而增加。相反,长期培养非癌性犬成纤维细胞或DNA修复缺陷型小鼠成纤维细胞(纯合子共济失调毛细血管扩张(ATM)基因缺陷)时,超中心染色体不会积累。总之,这些结果表明,亚中心染色体和端粒功能障碍是犬OSA的特征。因此,靶向这些独特的端粒畸变具有改善犬OSA的诊断以及开发新的治疗策略的潜力。最后,我们研究了代表不同癌症类型的犬癌细胞系中的放射敏感性。由不同肿瘤类型组成的人类癌细胞系NCI-60已成功用于体外抗癌药物筛选,证明这种方法在理解癌症机制中的实用性;无论肿瘤组织类型如何,基因表达谱分析和可变的放射敏感性都促进了信息标记的发展。最近在科罗拉多州立大学弗林特动物癌症中心(FACC)开发了一种称为ACC30的犬癌细胞系,其中包括微阵列基因表达数据。我们旨在检验这一假说,即在各种犬癌细胞系中确定对电离辐射的反应决定因素将提供其他信息,其中一些可能是狗特有的,而某些则可能是人类癌症报道的补充。我们从ACC30小组获得了来自十种肿瘤类型的27种犬细胞系。首先,对于克隆细胞培养使用克隆形成测定法,对于悬浮细胞培养使用有限稀释测定法来测定放射敏感性。无论肿瘤类型如何,这27种细胞系均具有不同的放射敏感性(2 Gy的存活分数,SF2 = 0.2--0.9)。根据本研究中分析的细胞特征,在27个细胞系中,抗辐射性与更好的铺板效率之间存在适度的相关性。接下来,我们选择放射线敏感性最高的六个细胞系作为放射敏感性组,选择放射线敏感性最高的五个细胞系作为放射敏感性组。然后,我们比较了辐射敏感性组与辐射敏感性组相比,通过电离辐射(IR)杀死细胞的已知参数,包括IR诱导的DNA双链断裂(DSB)修复和凋亡。但是,这两个组不能通过这些参数加以区分。此外,我们使用ACC面板针对20,000个基因的基础基因表达谱研究了一种可能的常见放射敏感性特征。鉴定出在放射敏感性和放射抗性基团之间差异表达的550多个基因。基因集富集分析被用于告知涉及差异表达基因的潜在途径和功能,实际上,包括细胞粘附,细胞迁移和凋亡在内的几种生物学过程与犬癌细胞系的放射敏感性有关。为了支持我们的发现,细胞粘附是先前在人类微阵列分析中确定的特征之一。总之,我们的结果表明,与细胞粘附相关的基因,而不是更普遍认为的放射敏感性相关的凋亡和DNA修复相关基因,可能为预测个体对放射治疗的反应提供有益的放射敏感性生物标志物,而与肿瘤类型无关。因此,此处表征的放射敏感性标记可以帮助指导内在肿瘤放射敏感性生物标记物在犬癌的预测分析中的未来发展,并且也有可能改善人类癌症的预测放射敏感性。

著录项

  • 作者

    Maeda, Junko.;

  • 作者单位

    Colorado State University.;

  • 授予单位 Colorado State University.;
  • 学科 Cellular biology.;Molecular biology.;Oncology.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 188 p.
  • 总页数 188
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:52:24

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