Hernández-Suárez Beatriz, Gillespie David A, Obmińska-Mrukowicz Bożena, Pawlak Aleksandra
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, 50-375 Wrocław, Poland.
Instituto de Tecnologías Biomédicas, Facultad de Medicina, Campus Ciencias de la Salud, Universidad de La Laguna, La Laguna 38071, Tenerife, Spain.
J Vet Res. 2023 Sep 20;67(3):447-458. doi: 10.2478/jvetres-2023-0042. eCollection 2023 Sep.
New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway.
The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine-murine and canine-human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death.
Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation.
These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.
犬类癌症患者急需新的、更有效的治疗方法，这就需要进行先进的实验研究。狗是比较肿瘤学研究的良好模型；然而，目前犬类癌细胞生物学研究受到有效抗体试剂和技术可用性低的限制。本研究使用市售抗体对一组造血犬癌细胞系中未折叠蛋白反应（UPR）关键成分的表达进行了表征，并验证了用于研究该途径的方法。
使用从外部获取的CLBL-1犬淋巴瘤细胞系和GL-1犬白血病细胞系以及在内部建立的两个对应细胞系（CNK-89和CLB70）作为不同淋巴瘤和白血病犬细胞系的模型进行研究。人U2OS细胞系作为对照。根据已知的犬细胞反应性以及犬-鼠和犬-人同源性选择用于鉴定UPR蛋白的抗体。在细胞系中用毒胡萝卜素和MG132诱导内质网应激。依托泊苷用于诱导细胞中的DNA损伤。用于该验证分析的技术包括RNA测序以观察犬细胞系中UPR成分的表达，蛋白质印迹以观察细胞诱导内质网应激后蛋白质表达水平的变化，以及流式细胞术以研究细胞死亡。
在犬癌细胞系中观察到UPR途径的基础表达和激动剂诱导激活方面存在显著差异，尽管这些差异的生物学意义需要进一步研究。
这些发现将成为未来犬类癌症生物学研究的起点。它们还将有助于为犬类患者开发新的抗癌疗法，并可能为人类肿瘤学提供新的见解。
