Name: an in vitro model for studying cellular transformation by kaposi sarcoma herpesvirus
Uploaded: 2017-08-25T12:30:00
Description: Watch this Scientific Journal Video about An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus at JoVE.com

This work was supported by K12 HD068322 (SCM); R01 CA179921 and P51 OD011092 (AVM); and award 14PRE20320014 from the America Heart Association (SB).

NOTE: All procedures described in this protocol should be performed under BSL-2 conditions.
1. KSHV Stock Preparation
<ol>
	<li>Prepare TNE buffer: dissolve 292.24 mg EDTA in ddH2O, bring to 225 mL, and adjust to pH 8. Dissolve 605.7 mg Tris in ddH2O, bring to 225 mL, and adjust to pH 8. Combine EDTA and Tris solutions, add 4.38 g NaCl, adjust final volume to 500 mL, filter sterilize, and store at 4 &#176;C.</li>
	<li>Culture the KSHV-positive, EBV-negative p...

Oncogenesis is a multistep process that circumvents important safeguards within an organism36. As KS lesions exist along a spectrum of chronic inflammation to true sarcomas, elucidation of certain pathophysiological processes mediated by KSHV requires that some studies be conducted in cell culture models that support transformation9. It should be noted that loss of contact inhibition and anchorage-dependent growth, phenotypes indicative of cellular transformation, do not re...

Kaposi sarcoma (KS) is a multi-focal angioproliferative tumor affecting dermal, mucosal, and visceral sites that develops most commonly in the setting of advanced immune suppression1. Four epidemiological forms have been described: classic, an indolent form that typically affects older people of Mediterranean and Middle Eastern heritage; iatrogenic, resulting from treatment with immunosuppressive drugs following organ transplantation; epidemic, an AIDS-defining cancer; and endemic, an HIV-independent form common in children in endemic regions in Africa. With the advent of effective combination anti-retroviral drug regimens for the treatment of HIV, epidemic KS is much less commonly diagnosed in developing countries. However, the clinically aggressive endemic and epidemic forms remain among the most commonly diagnosed cancers in many African countries2,3,4. Therefore, identification of effective pathogenesis-targeted drugs for treatment of KS is a research priority.
Histologically, KS lesions are characterized by extensive but abnormal neovascularization whereby spindle cells of EC origin form discontinuous vascular networks5. These abnormal vessels (&#34;vascular slits&#34;) allow extravasation of erythrocytes, which give lesions their characteristic color. Additionally, lesions contain numerous leukocytes that characterize chronic inflammation (i.e., lymphocytes, macrophages, and plasma cells). Regression of KS lesions following immune reconstitution has been described, suggesting that KS has features of both a hyper-proliferative lesion and a true tumor6,7,8,9.
KS herpesvirus (KSHV), the causative agent of KS, was identified in 199410. Since then many in vitro cell culture models have been developed to enable pathogenesis studies, including cells explanted from tumor biopsy material and primary or telomerase-expressing EC infected with KSHV in vitro11,12,13,14,15,16,17,18. None of the currently available models fully recapitulates the KS tumor microenvironment, but all have contributed valuable knowledge to our understanding of the pathobiology of KSHV infection. Unlike the other known tumorigenic human herpesvirus Epstein-Barr virus (EBV), KSHV does not readily transform cells in culture following de novo infection19,20,21,22. However, this limitation has been overcome by transducing primary human EC of either mixed microvascular or lymphatic origin with the E6 and E7 genes from human papillomavirus type 16 prior to infection with KSHV23,24. Expression of these exogenous oncogenes dramatically increases the transforming potential of KSHV in vitro in part by providing further inhibition of the retinoblastoma protein and p5323,24. This EC transduction method has allowed multiple laboratories to identify key alterations in host cell gene expression that are induced by KSHV infection and that appear to facilitate KS cell survival and proliferation25,26,27,28,29,30,31,32. The protocols described herein are straightforward and highly reproducible, and will result in the generation of age- and passage-matched KSHV-infected EC and mock-infected controls that can be cultured for far longer than primary cells and will allow for the investigation of oncogenic mechanisms employed by KSHV. Although the protocol includes a method for production of wild type KSHV from the primary effusion lymphoma cell line BCBL-1, E6/E7-immortalized EC are also highly susceptible to infection with recombinant BACmid derived KSHV-BAC1630. Protocols for preparation of BAC16 are described elsewhere33,34.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCBL-1 cells</td>
			<td>NIH AIDS Reagent Program</td>
			<td>3233</td>
		</tr>
		<tr>
			<td>PA317 cells</td>
			<td>ATCC</td>
			<td>CRL-2203</td>
		</tr>
		<tr>
			<td>Neonatal dermal microvascular endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2505</td>
		</tr>
		<tr>
			<td>EBM-2 Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
		</tr>
		<tr>
			<td>EGM-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
		</tr>
		<tr>
			<td>anti-KSHV LANA/ORF 73</td>
			<td>Advanced Biotechnologies</td>
			<td>13-210-100</td>
		</tr>
		<tr>
			<td>TrypLETMExpress, no phenol red</td>
			<td>ThermoFisher</td>
			<td>12604013</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model for studying cellular transformation by kaposi sarcoma herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

The morphology of primary EC is classically described as &#34;cobble stone&#34;, and this morphology is not altered by expression of the papillomavirus E6 and E7 genes (Figure 1A). Expression of the E6 and E7 genes alone does not induce a transformed phenotype; thus, cells are susceptible to contact inhibition and will cease dividing upon reaching confluence in culture. The cells will however proliferate and regrow to confluence upon trypsinization and replating at a lowe...

Watch this Scientific Journal Video about An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus at JoVE.com

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

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