JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Biologie

一种用于研究卡波西肉瘤细胞转化的体外模型

Published: August 25, 2017
doi:
10.3791/54828
, , , ,
1Division of Pediatric Infectious Diseases,University of Minnesota Medical School, 2Vaccine and Gene Therapy Institute,Oregon Health and Science University

Summary

卡波西肉瘤 (KS) 是一种由感染致癌病毒的人 herpesvirus-8/KS 疱疹 (HHV-8/KSHV) 引起的肿瘤。这里描述的内皮细胞培养模型是唯一适合研究 KSHV 转化宿主细胞的机制。

Abstract

卡波西肉瘤 (KS) 是一种不寻常的肿瘤, 由内皮细胞 (EC) 与 KSHV 感染引起的梭形细胞组成, 在免疫抑制的设置中最常见。尽管进行了几十年的研究, 但对 KS 的最佳治疗仍未明确, 临床结果在资源有限的环境中尤其不利。KS 病变是由病理血管生成, 慢性炎症, 和发生, 和各种体外细胞培养模型的发展, 以研究这些过程。KS 产生于 KSHV 感染的内皮细胞, 因此 EC 谱系细胞提供最合适的体外的纺锤细胞前体的代孕。然而, 由于 EC 有一个有限的体外寿命, 而且由于 KSHV 所采用的致癌机制比其他瘤病毒的效率低, 因此很难评估初级或端粒酶-永生 EC。因此, 建立了一种新的 EC-based 培养模型, 可随时支持 KSHV 感染后的转化。人 E6 和 E7 基因的异位表达16型, 允许延长年龄和通道匹配的模拟和 KSHV 感染 EC 的培养, 并支持在受感染细胞培养中真正转变的 (即瘤) 表型的发展.这种易于处理和高度重现的 KS 模型, 有助于发现一些重要的信号通路, 具有很高的潜力, 翻译成临床设置。

Introduction

卡波西肉瘤 (KS) 是一种多的 angioproliferative 肿瘤, 影响真皮, 黏膜, 和内脏的网站, 发展最常见的设置先进的免疫抑制1。四流行病学形式被描述了: 典型, 懒惰形式典型地影响地中海和中东遗产的老年人;医源性, 由器官移植后免疫抑制剂治疗所致;流行病, 一种艾滋病定义的癌症;和地方性流行病, 是非洲流行地区儿童常见的一种艾滋病毒独立形式。随着有效的抗逆转录病毒药物治疗方案的问世, 艾滋病毒的流行在发展中国家被普遍诊断为不多。然而, 在许多非洲国家, 临床上侵袭性流行和流行的形式仍然是最常见的诊断癌症2,3,4。因此, 确定有效的病机-靶向药物治疗 KS 是一个研究的重点。

组织学上, KS 病变的特点是广泛的, 但不正常的新生血管, 即纺锤细胞的 EC 起源形成不连续的维管网络5。这些异常血管 (“血管狭缝”) 允许红细胞外渗, 从而使病灶具有其特有的颜色。此外, 病灶中含有大量的白细胞, 它们是慢性炎症的特征 (如淋巴细胞、巨噬细胞和血浆电池)。免疫重建后 ks 病变的回归已被描述, 这表明 ks 具有超增生性病变和真正的肿瘤的特点6,7,8,9。

ks 疱疹病毒 (KSHV), 致病剂 ks, 被确定在 1994年10。自那时以来, 许多体外细胞培养模型已经开发, 以使发病机制的研究, 包括细胞说明从肿瘤活检材料和主要或端粒酶表达 EC 感染 KSHV体外11,12,13,14,15,16,17,18. 目前尚无可用的模型完全重述了 KS 肿瘤微环境, 但都为我们了解 KSHV 感染的病理提供了宝贵的知识。不像其他已知的瘤人类疱疹病毒-巴尔 (eb), KSHV 不容易转换在文化中的细胞后de 从头感染19,20,21, 22. 然而, 这一限制已经克服了换初级人类 EC 的混合微血管或淋巴来源与 E6 和 E7 基因的人 hpv 类型16之前感染 KSHV23,24.这些外源癌基因的表达显著增加了 KSHV 的转化潜能在体外部分通过进一步抑制视网膜母细胞瘤蛋白和 p5323,24。这种 EC 转导方法允许多个实验室识别 KSHV 感染诱发的宿主细胞基因表达的关键改变, 并显示出促进 KS 细胞存活和增殖的作用25,26,27,28,29,30,31,32. 本文所述的协议是直接的和高度可重现的, 并将导致产生年龄和通道匹配的 KSHV 感染 EC 和模拟感染的控制, 可以培养远长于原细胞, 并将允许调查 KSHV 所采用的致癌机制。虽然该协议包括一种从原发性渗出性淋巴瘤细胞系 BCBL-1 中生产野生型 KSHV 的方法, 但 E6/E7-immortalized EC 也很容易感染重组 BACmid 源性 KSHV-BAC1630。在其他地方33,34中描述了用于准备 BAC16 的协议。

Protocol

注意: 本协议中描述的所有过程都应在 BSL-2 条件下执行. 1. KSHV 库存准备 准备缓冲区: 在 ddH 2 O 中溶解292.24 毫克 EDTA, 带到225毫升, 并调整到 pH 8。溶解605.7 毫克在 ddH 2 O, 带来225毫升, 并调整到 pH 值8。结合 EDTA 和三磷酸溶液, 加入4.38 克氯化钠, 调整最终体积到500毫升, 过滤杀菌, 并存储在4和 #176; C . 培养 KSHV 阳性、eb 病毒阴性的?…

Representative Results

原 EC 的形态学被经典地描述为 “鹅卵石”, 这种形态学不被 E6 和 E7 基因 (图 1A) 的表达所改变。单 E6 和 E7 基因的表达不诱导转化表型;因此, 细胞易于接触抑制, 并将停止分裂后, 达到融合的文化。然而, 细胞将增殖和再生的汇合后, trypsinization 和 replating 在较低的密度, 允许维护年龄和通道匹配的文化, 作为模拟感染的控制使用。 <p class="jove_content" fo:keep-together….

Discussion

发生是一个多步骤的过程, 它绕过了一个有机体36中的重要安全措施。由于 KS 病变存在于慢性炎症的频谱, 以真正的肉瘤, 阐明某些病理生理学过程介导的 KSHV 要求, 一些研究在细胞培养模式, 支持转换9。应该指出的是, 接触抑制的丧失和锚定依赖性生长, 表型指示细胞转化, 不容易发展继发于原 ec 或 ec 的外源表达的端粒酶的感染。因此, 虽然在这里描述的 ks 的<em…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 K12 HD068322 (SCM) 的支持;R01 CA179921 和 P51 OD011092 (AVM);并授予美国心脏协会 (SB) 14PRE20320014 奖。

Materials

BCBL-1 cells NIH AIDS Reagent Program 3233
PA317 cells ATCC CRL-2203
Neonatal dermal microvascular endothelial cells Lonza CC-2505
EBM-2 Basal Medium Lonza CC-3156
EGM-2 BulletKit Lonza CC-3162
anti-KSHV LANA/ORF 73 Advanced Biotechnologies 13-210-100
TrypLETMExpress, no phenol red ThermoFisher 12604013
RPMI
DMEM
PBS with calcium and magnesium
DOWNLOAD MATERIALS LIST

References

  1. Bhutani, M., Polizzotto, M. N., Uldrick, T. S., Yarchoan, R. Kaposi sarcoma-associated herpesvirus-associated malignancies: epidemiology, pathogenesis, and advances in treatment. Sem. Onc. 42 (2), 223-246 (2015).
  2. Dedicoat, M., Vaithilingum, M., Newton, R. Treatment of Kaposi’s sarcoma in HIV-1 infected individuals with emphasis on resource poor settings. The Cochrane database. (3), CD003256 (2003).
  3. Robey, R. C., Bower, M. Facing up to the ongoing challenge of Kaposi’s sarcoma. Curr. Op. Inf. Dis. 28 (1), 31-40 (2015).
  4. Sasco, A. J., et al. The challenge of AIDS-related malignancies in sub-Saharan Africa. PLOS ONE. 5 (1), e8621 (2010).
  5. Grayson, W., Pantanowitz, L. Histological variants of cutaneous Kaposi sarcoma. Diag. Path. 3, 31 (2008).
  6. Cattelan, A. M., et al. Regression of AIDS-related Kaposi’s sarcoma following antiretroviral therapy with protease inhibitors: biological correlates of clinical outcome. Euro. J. Can. 35 (13), 1809-1815 (1999).
  7. Yuan, D., et al. Use of X-Chromosome Inactivation Pattern to Analyze the Clonality of 14. Female Cases of Kaposi Sarcoma. Med. Sci. Mon. Bas. Res. 21, 116-122 (2015).
  8. Gill, P. S., et al. Evidence for multiclonality in multicentric Kaposi’s sarcoma. PNAS. 95 (14), 8257-8261 (1998).
  9. Douglas, J. L., Gustin, J. K., Moses, A. V., Dezube, B. J., Pantanowitz, L. Kaposi Sarcoma Pathogenesis: A Triad of Viral Infection, Oncogenesis and Chronic Inflammation. Trans. Biomed. 1 (2), (2010).
  10. Chang, Y., et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science. 266 (5192), 1865-1869 (1994).
  11. Moses, A. In vitro endothelial cell systems to study Kaposi sarcoma. Kaposi Sarcoma: A Model of Oncogenesis. (Chapter 4), (2010).
  12. McAllister, S. C., Moses, A. V. Endothelial Cell- and Lymphocyte-Based In Vitro Systems for Understanding KSHV Biology. Kaposi Sarcoma Herpesvirus: New Perspectives. 312 (Chapter 8), 211-244 (2007).
  13. Lagunoff, M., et al. De Novo Infection and Serial Transmission of Kaposi’s Sarcoma-Associated Herpesvirus in Cultured Endothelial Cells. J. Virol. 76 (5), 2440-2448 (2002).
  14. Benelli, R., Repetto, L., Carlone, S., Parravicini, C. Establishment and characterization of two new Kaposi’s sarcoma cell cultures from an AIDS and a non-AIDS patient. Res. Virol. 145, 251-259 (1994).
  15. Simonart, T., et al. Iron as a potential co-factor in the pathogenesis of Kaposi’s sarcoma?. Int. J. Can. 78 (6), 720-726 (1998).
  16. An, F. Q., et al. Long-Term-Infected Telomerase-Immortalized Endothelial Cells: a Model for Kaposi’s Sarcoma-Associated Herpesvirus Latency In Vitro and In Vivo. J. Virol. 80 (10), 4833-4846 (2006).
  17. Myoung, J., Ganem, D. Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction. J. Virol. Meth. 174 (1-2), 12-21 (2011).
  18. Lunardi-lskandar, Y., et al. Tumorigenesis and metastasis of neoplastic Kaposi’s sarcoma cell line in immunodeficient mice blocked by a human pregnancy hormone. Nature. 375 (6526), 64-68 (1995).
  19. Cesarman, E. Gammaherpesvirus and lymphoproliferative disorders in immunocompromised patients. Can. Let. 305 (2), 163-174 (2011).
  20. Jones, T., et al. Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV. J. Clin. Inves. 122 (3), 1076-1081 (2012).
  21. Aguirre, A. J., Robertson, E. S. Epstein-Barr Virus Recombinants from BC-1 and BC-2 Can Immortalize Human Primary B Lymphocytes with Different Levels of Efficiency and in the Absence of Coinfection by Kaposi’s Sarcoma-Associated Herpesvirus. J. Virol. 74 (2), 735-743 (2000).
  22. Flore, O., et al. Transformation of primary human endothelial cells by Kaposi’s sarcoma-associated herpesvirus. Nature. 394 (6693), 588-592 (1998).
  23. Moses, A. V., et al. Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8. J. Virol. 73 (8), 6892-6902 (1999).
  24. Halbert, C. L., Demers, G. W., Galloway, D. A. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65 (1), 473-478 (1991).
  25. Moses, A. V., et al. Kaposi’s Sarcoma-Associated Herpesvirus-Induced Upregulation of the c-kit Proto-Oncogene, as Identified by Gene Expression Profiling, Is Essential for the Transformation of Endothelial Cells. J. Virol. 76 (16), 8383-8399 (2002).
  26. McAllister, S. C., et al. Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells. Blood. 103 (9), 3465-3473 (2004).
  27. Raggo, C., et al. Novel cellular genes essential for transformation of endothelial cells by Kaposi’s sarcoma-associated herpesvirus. Can. Res. 65 (12), 5084-5095 (2005).
  28. McAllister, S. C., Hanson, R. S., Manion, R. D. Propranolol Decreases Proliferation of Endothelial Cells Transformed by Kaposi’s Sarcoma-Associated Herpesvirus and Induces Lytic Viral Gene Expression. J. Virol. 89 (21), 11144-11149 (2015).
  29. Rose, P. P., Bogyo, M., Moses, A. V., Früh, K. Insulin-like growth factor II receptor-mediated intracellular retention of cathepsin B is essential for transformation of endothelial cells by Kaposi’s sarcoma-associated herpesvirus. J. Virol. 81 (15), 8050-8062 (2007).
  30. Botto, S., Totonchy, J. E., Gustin, J. K., Moses, A. V. Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms. mBio. 6 (3), e00668 (2015).
  31. Mansouri, M. Kaposi sarcoma herpesvirus K5 removes CD31/PECAM from endothelial cells. Blood. 108 (6), 1932-1940 (2006).
  32. Mansouri, M., Rose, P. P., Moses, A. V., Fruh, K. Remodeling of Endothelial Adherens Junctions by Kaposi’s Sarcoma-Associated Herpesvirus. J. Virol. 82 (19), 9615-9628 (2008).
  33. Brulois, K. F., et al. Construction and manipulation of a new Kaposi’s sarcoma-associated herpesvirus bacterial artificial chromosome clone. J. Virol. 86 (18), 9708-9720 (2012).
  34. Myoung, J., Ganem, D. Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction. J. Virol. Meth. 174 (1-2), 12-21 (2011).
  35. Prudhomme, J. G., Sherman, I. W., Land, K. M. Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells. Int. J. Parasit. 26 (6), 647-655 (1996).
  36. Hanahan, D., Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell. 144 (5), 646-674 (2011).
  37. Walker, L. R., Hussein, H. A. M., Akula, S. M. Disintegrin-like domain of glycoprotein B regulates Kaposi’s sarcoma-associated herpesvirus infection of cells. J. Gen. Virol. 95 (Pt 8), 1770-1782 (2014).
  38. Dai, L., et al. Sphingosine Kinase-2 Maintains Viral Latency and Survival for KSHV-Infected Endothelial Cells. PLOS ONE. 9 (7), e102314-e102319 (2014).
  39. Yoo, J., et al. Opposing Regulation of PROX1 by Interleukin-3 Receptor and NOTCH Directs Differential Host Cell Fate Reprogramming by Kaposi Sarcoma Herpes Virus. PLoS Path. 8 (6), e1002770 (2012).
  40. Sharma-Walia, N., et al. COX-2/PGE2: molecular ambassadors of Kaposi’s sarcoma-associated herpes virus oncoprotein-v-FLIP. Oncogenesis. 1 (4), e5 (2012).
  41. Dimaio, T. A., Gutierrez, K. D., Lagunoff, M. Latent KSHV Infection of Endothelial Cells Induces Integrin Beta3 to Activate Angiogenic Phenotypes. PLoS Path. 7 (12), e1002424 (2011).
  42. Wu, Y. H., et al. The manipulation of miRNA-gene regulatory networks by KSHV induces endothelial cell motility. Blood. 118 (10), 2896-2905 (2011).
  43. Alcendor, D. J., Knobel, S., Desai, P., Zhu, W. Q., Hayward, G. S. KSHV Regulation of Fibulin-2 in Kaposi’s Sarcoma: implications for tumorigenesis. Am. J. Path. 179 (3), 1443-1454 (2011).
  44. Poole, L. J., et al. Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi’s Sarcoma-Associated Herpesvirus. J. Virol. 76 (7), 3395-3420 (2002).
  45. Toth, Z., et al. Epigenetic Analysis of KSHV Latent and Lytic Genomes. PLoS Path. 6 (7), e1001013 (2010).
  46. Damania, B., et al. Comparison of the Rta/Orf50 transactivator proteins of gamma-2-herpesviruses. J. Virol. 78 (10), 5491-5499 (2004).
  47. Koon, H. B., et al. Phase II trial of imatinib in AIDS-associated Kaposi’s sarcoma: AIDS Malignancy Consortium Protocol 042. J. Clin. Onc. 32 (5), 402-408 (2014).
  48. Koon, H. B., et al. Imatinib-induced regression of AIDS-related Kaposi’s sarcoma. J. Clin. Onc. 23 (5), 982-989 (2005).
  49. Cao, W., et al. Imatinib for highly chemoresistant Kaposi sarcoma in a patient with long-term HIV control: a case report and literature review. Cur. Onc. 22 (5), 395 (2015).
  50. Botto, S., Totonchy, J. E., Gustin, J. K., Moses, A. V. Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms. mBio. 6 (3), e00668 (2015).
  51. Pantanowitz, L., et al. C-Kit (CD117) expression in AIDS-related, classic, and African endemic Kaposi sarcoma. App. Imm. & Mol. Morph. 13 (2), 162-166 (2005).
  52. Poole, L. J., et al. Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi’s Sarcoma-Associated Herpesvirus. J. Virol. 76 (7), 3395-3420 (2002).
  53. Desnoyer, A., et al. Expression pattern of the CXCL12/CXCR4-CXCR7 trio in Kaposi sarcoma skin lesions. Brit. J. Derm. , 1-12 (2016).
  54. Dai, L., et al. Role of heme oxygenase-1 in the pathogenesis and tumorigenicity of Kaposi’s sarcoma-associated herpesvirus. Oncotarget. 7 (9), 10459-10471 (2016).
  55. Chisholm, K. M., et al. β-Adrenergic receptor expression in vascular tumors. Modern Pathology. 25 (11), 1446-1451 (2012).
  56. Gerber, H. P., et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem. 273 (46), 30336-30343 (1998).
  57. Billstrom Schroeder, ., Christensen, M. R., Worthen, G. S. Human cytomegalovirus protects endothelial cells from apoptosis induced by growth factor withdrawal. J. Clin. Virol. 25 (Suppl 2), S149-S157 (2002).

Tags

In Vitro ModelCellular TransformationKaposi Sarcoma HerpesvirusKSHVEndothelial CellsMalignant TransformationViral OncogenesisKaposi SarcomaPathological AngiogenesisBCBL-1 CellsPMAViral Particle IsolationUltracentrifugation
check_url/fr/54828?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
McAllister, S. C., Hanson, R. S., Grissom, K. N., Botto, S., Moses, A. V. An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus. J. Vis. Exp. (126), e54828, doi:10.3791/54828 (2017).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image