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• W1978855492 abstract "Transfection of a Kaposi's sarcoma (KS) herpesvirus (KSHV) Bacterial Artificial Chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells generates a cell (mECK36) that forms KS-like tumors in mice. mECK36 expressed most KSHV genes and were angiogenic, but they didn't form colonies in soft agar. In nude mice, mECK36 formed KSHV-harboring vascularized spindle cell sarcomas that were LANA+/podoplanin+, overexpressed VEGF and Angiopoietin ligands and receptors, and displayed KSHV and host transcriptomes reminiscent of KS. mECK36 that lost the KSHV episome reverted to nontumorigenicity. siRNA suppression of KSHV vGPCR, an angiogenic gene upregulated in mECK36 tumors, inhibited angiogenicity and tumorigenicity. These results show that KSHV malignancy is in vivo growth restricted and reversible, defining mECK36 as a biologically sensitive animal model of KSHV-dependent KS. Transfection of a Kaposi's sarcoma (KS) herpesvirus (KSHV) Bacterial Artificial Chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells generates a cell (mECK36) that forms KS-like tumors in mice. mECK36 expressed most KSHV genes and were angiogenic, but they didn't form colonies in soft agar. In nude mice, mECK36 formed KSHV-harboring vascularized spindle cell sarcomas that were LANA+/podoplanin+, overexpressed VEGF and Angiopoietin ligands and receptors, and displayed KSHV and host transcriptomes reminiscent of KS. mECK36 that lost the KSHV episome reverted to nontumorigenicity. siRNA suppression of KSHV vGPCR, an angiogenic gene upregulated in mECK36 tumors, inhibited angiogenicity and tumorigenicity. These results show that KSHV malignancy is in vivo growth restricted and reversible, defining mECK36 as a biologically sensitive animal model of KSHV-dependent KS. Kaposi's sarcoma (KS) herpesvirus (KSHV) is the etiologic agent of KS, an angiogenic spindle cell sarcoma associated with AIDS. The mechanisms driving KSHV carcinogenesis could be uncovered through reproduction of KS by KSHV infection of normal cells. Our results defining this cell and animal model of KS show that KSHV-induced neoplasia is reversible, KSHV dependent, and in vivo restricted. Upregulation of KSHV genes, such as the proangiogenic vGPCR, under in vivo growth conditions provides a selective advantage to KSHV-harboring cells and leads to episome maintenance and tumorigenesis. Thus, mECK36 tumors are phenotypic, molecular, and viral surrogates of KS that are suitable for analyzing the role of viral and host genes in KS pathogenesis, and for preclinical testing of anti-KS drugs. Human herpesvirus-8 or Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three AIDS-related malignancies: Kaposi's sarcoma (KS) (Boshoff and Weiss, 1998Boshoff C. Weiss R.A. Kaposi's sarcoma-associated herpesvirus.Adv. Cancer Res. 1998; 75: 57-86Crossref PubMed Google Scholar, Chang et al., 1994Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D. Moore P. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposis's sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4744) Google Scholar, Ganem, 2006Ganem D. KSHV infection and the pathogenesis of Kaposi's sarcoma.Annu. Rev. Pathol. Mech. Dis. 2006; 1: 273-296Crossref PubMed Scopus (227) Google Scholar), Multicentric Castlemans disease (Dupin et al., 1999Dupin N. Fisher C. Kellam P. Ariad S. Tulliez M. Franck N. van Marck E. Salmon D. Gorin I. Escande J.P. et al.Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.Proc. Natl. Acad. Sci. USA. 1999; 96: 4546-4551Crossref PubMed Scopus (595) Google Scholar), and primary effusion lymphoma (Cesarman et al., 1995Cesarman E. Chang Y. Moore P.S. Said J.W. Knowles D.M. Kaposi's sarcoma-associated herpes virus-like DNA sequences are present in AIDS-related body cavity B-cell lymphomas.N. Engl. J. Med. 1995; 332: 1186-1191Crossref PubMed Scopus (2408) Google Scholar). In HIV-infected individuals, predominantly in the male homosexual population, the incidence of KS dramatically increases and can manifest as an advanced disseminated cancer with increased morbidity and mortality (Gallo, 1998Gallo R.C. The enigmas of Kaposi's sarcoma.Science. 1998; 282: 1837-1839Crossref PubMed Google Scholar). The development of pathogenesis-based therapies is important for improving current KS treatments (Pantanowitz and Dezube, 2004Pantanowitz L. Dezube B.J. Advances in the pathobiology and treatment of Kaposi sarcoma.Curr. Opin. Oncol. 2004; 16: 443-449Crossref PubMed Scopus (37) Google Scholar). KS presents itself as multifocal lesions in the skin, lungs, and gastrointestinal tract (Safai et al., 1985Safai B. Johnson K.G. Myskowski P.L. Koziner B. Yang S.Y. Winningham-Ruddles S. Godbold J.H. Dupont B. The natural history of Kaposi's sarcoma in Acquired Immunodeficiency Syndrome.Ann. Intern. Med. 1985; 103: 744-750Crossref PubMed Scopus (209) Google Scholar). It is characterized by intense VEGF-mediated angiogenesis, spindle cell proliferation, and erythrocyte extravasation (Gallo, 1998Gallo R.C. The enigmas of Kaposi's sarcoma.Science. 1998; 282: 1837-1839Crossref PubMed Google Scholar, Safai et al., 1985Safai B. Johnson K.G. Myskowski P.L. Koziner B. Yang S.Y. Winningham-Ruddles S. Godbold J.H. Dupont B. The natural history of Kaposi's sarcoma in Acquired Immunodeficiency Syndrome.Ann. Intern. Med. 1985; 103: 744-750Crossref PubMed Scopus (209) Google Scholar). There are many key unanswered issues in KS pathogenesis that are matters of controversy and intense research: (1) the neoplastic nature of KS, (2) the identity of the normal cell type that upon infection with KSHV becomes a malignant KS spindle cell, (3) the connection between KSHV gene expression and the KS phenotype, and (4) the relationship between KSHV biology and other KS cofactors, such as immunosuppression, HIV infection, and sexual transmission. Primary to answering these questions is the development of an experimental model of KSHV infection that reproduces the main pathogenic phenotypes of KS in vitro and in animals. While spindle cells are generally considered the tumor cell in KS lesions, their origin and true malignant nature are controversial, because they lack many features of neoplastic cells such as aneuploidy, tumorigenicity (Gallo, 1998Gallo R.C. The enigmas of Kaposi's sarcoma.Science. 1998; 282: 1837-1839Crossref PubMed Google Scholar), or clonality (Gill et al., 1998Gill P.S. Tsai Y.C. Rao A.P. Spruck 3rd, C.H. Zheng T. Harrington Jr., W.A. Cheung T. Nathwani B. Jones P.A. Evidence for multiclonality in multicentric Kaposi's sarcoma.Proc. Natl. Acad. Sci. USA. 1998; 95: 8257-8261Crossref PubMed Scopus (110) Google Scholar). KS spindle cells express phenotypic markers that belong to many cell lineages, including endothelium, smooth muscle cells, macrophages, and hematopoietic cells (Boshoff and Weiss, 2002Boshoff C. Weiss R. AIDS-related malignancies.Nat. Rev. Cancer. 2002; 2: 373-382Crossref PubMed Scopus (297) Google Scholar, Gallo, 1998Gallo R.C. The enigmas of Kaposi's sarcoma.Science. 1998; 282: 1837-1839Crossref PubMed Google Scholar). Since KS could present itself as a multifocal disease, and KS spindle cells express the hematopoietic stem cell marker CD34, it has been proposed that the KS progenitor cell is a circulating cell of the hematopoietic endothelial lineage (Barozzi et al., 2003Barozzi P. Luppi M. Facchetti F. Mecucci C. Alu M. Sarid R. Rasini V. Ravazzini L. Rossi E. Festa S. et al.Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors.Nat. Med. 2003; 9: 554-561Crossref PubMed Scopus (179) Google Scholar, Boshoff and Weiss, 2002Boshoff C. Weiss R. AIDS-related malignancies.Nat. Rev. Cancer. 2002; 2: 373-382Crossref PubMed Scopus (297) Google Scholar, Browning et al., 1994Browning P.J. Sechler J.M.G. Kaplan M. Washington R.H. Gendelman R. Yarchoan R. Ensoli B. Gallo R.C. Identification and culture of Kaposi's sarcoma-like spindle cells from the peripheral blood of human immunodeficiency virus-1 individualas and normal controls.Blood. 1994; 84: 2711-2720PubMed Google Scholar). Spindle cells in the lesions express markers characteristic of the lymphatic endothelial cell (EC) lineage, such as LYVE-1, podoplanin, and VEGF-R3. It has been recently suggested that they originate from lymphatic endothelium (Dupin et al., 1999Dupin N. Fisher C. Kellam P. Ariad S. Tulliez M. Franck N. van Marck E. Salmon D. Gorin I. Escande J.P. et al.Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.Proc. Natl. Acad. Sci. USA. 1999; 96: 4546-4551Crossref PubMed Scopus (595) Google Scholar, Skobe et al., 1999Skobe M. Brown L.F. Tognazzi K. Ganju R.K. Dezube B.J. Alitalo K. Detmar M. Vascular endothelial growth factor-C (VEGF-C) and its receptors KDR and flt-4 are expressed in AIDS-associated Kaposi's sarcoma.J. Invest. Dermatol. 1999; 113: 1047-1053Crossref PubMed Scopus (106) Google Scholar), transdifferentiated vascular endothelium, or a common EC precursor (Hong et al., 2004Hong Y.K. Foreman K. Shin J.W. Hirakawa S. Curry C.L. Sage D.R. Libermann T. Dezube B.J. Fingeroth J.D. Detmar M. Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.Nat. Genet. 2004; 36: 683-685Crossref PubMed Scopus (303) Google Scholar, Wang et al., 2004Wang H.W. Trotter M.W. Lagos D. Bourboulia D. Henderson S. Makinen T. Elliman S. Flanagan A.M. Alitalo K. Boshoff C. Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma.Nat. Genet. 2004; 36: 687-693Crossref PubMed Scopus (380) Google Scholar). It is now well established that KSHV is an etiologic cofactor strictly necessary for KS (Boshoff and Weiss, 1998Boshoff C. Weiss R.A. Kaposi's sarcoma-associated herpesvirus.Adv. Cancer Res. 1998; 75: 57-86Crossref PubMed Google Scholar, Chang et al., 1994Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D. Moore P. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposis's sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4744) Google Scholar, Ganem, 2006Ganem D. KSHV infection and the pathogenesis of Kaposi's sarcoma.Annu. Rev. Pathol. Mech. Dis. 2006; 1: 273-296Crossref PubMed Scopus (227) Google Scholar), indicating that KSHV genetic expression is responsible for the KS angiogenic phenotype. Although the KSHV genome encodes for genes that can induce cell transformation (Gao et al., 1997Gao S.J. Boshoff C. Jayachandra S. Weiss R.A. Chang Y. Moore P.S. KSHV ORF K9 (vIRF) is an oncogene which inhibits the interferon signaling pathway.Oncogene. 1997; 15: 1979-1985Crossref PubMed Scopus (311) Google Scholar, Lee et al., 1998Lee H. Veazey R. Williams K. Li M. Guo J. Neipel F. Fleckenstein B. Lackner A. Desrosiers R.C. Jung J.U. Deregulation of cell growth by the K1 gene of Kaposi's sarcoma-associated herpesvirus.Nat. Med. 1998; 4: 435-440Crossref PubMed Scopus (252) Google Scholar, Wang et al., 2006Wang L. Dittmer D.P. Tomlinson C.C. Fakhari F.D. Damania B. Immortalization of primary endothelial cells by the K1 protein of Kaposi's sarcoma-associated herpesvirus.Cancer Res. 2006; 66: 3658-3666Crossref PubMed Scopus (118) Google Scholar), immune deregulation (Moore et al., 1996Moore P.S. Boshoff C. Weiss R.A. Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by kshv.Science. 1996; 274: 1739-1744Crossref PubMed Scopus (800) Google Scholar, Nicholas et al., 1997Nicholas J. Ruvolo V.R. Burns W.H. Sandford G. Wan X.Y. Ciufo D. Hendrickson S.B. Guo H.G. Hayward G.S. Reitz M.S. Kaposis sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inflammatory protein-1 and interleukin-6.Nat. Med. 1997; 3: 287-292Crossref PubMed Scopus (326) Google Scholar), and angiogenesis activation (Aoki et al., 1999Aoki Y. Jaffe E.S. Chang Y. Jones K. Teruya-Feldstein J. Moore P.S. Tosato G. Angiogenesis and hematopoiesis induced by Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6.Blood. 1999; 93: 4034-4043Crossref PubMed Google Scholar, Bais et al., 1998Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.Nature. 1998; 391: 86-89Crossref PubMed Scopus (726) Google Scholar, Bais et al., 2003Bais C. Van Geelen A. Eroles P. Mutlu A. Chiozzini C. Dias S. Silverstein R. Rafii S. Mesri E.A. Kaposi's sarcoma associated herpesvirus G protein coupled receptor immortalizes human endothelial cell by activation of the VEGF receptor-2/KDR.Cancer Cell. 2003; 3: 131-143Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Boshoff et al., 1997Boshoff C. Endo Y. Collins P.D. Takeuchi Y. Reeves J.D. Schweickart V.L. Siani M.A. Sasaki T. Williams T.J. Gray P.W. et al.Angiogenic and HIV-inhibitory functions of KSHV-encoded chemokines.Science. 1997; 278: 290-294Crossref PubMed Scopus (432) Google Scholar, Montaner et al., 2003Montaner S. Sodhi A. Molinolo A. Bugge T.H. Sawai E.T. He Y. Li Y. Ray P.E. Gutkind J.S. Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes.Cancer Cell. 2003; 3: 23-36Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, Yang et al., 2000Yang T. Chen S. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C. Narula S. Chensue S. Lira S.A. Transgenic expression of the chemokine receptor encoded by HHV8 induces an angioprolierative disease resembling Kaposi's sarcoma.J. Exp. Med. 2000; 191: 445-454Crossref PubMed Scopus (353) Google Scholar), KSHV infection leads to KS in limited circumstances. Since KSHV is an endothelial-tropic virus, several KSHV-infection models using ECs have been described (Ciufo et al., 2001Ciufo D.M. Cannon J.S. Poole L.J. Wu F.Y. Murray P. Ambinder R.F. Hayward G.S. Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures.J. Virol. 2001; 75: 5614-5626Crossref PubMed Scopus (155) Google Scholar, Flore et al., 1998Flore O. Rafii S. Ely S. O'Leary J.J. Hyjek E.M. Cesarman E. Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.Nature. 1998; 394: 588-592Crossref PubMed Scopus (341) Google Scholar, Hong et al., 2004Hong Y.K. Foreman K. Shin J.W. Hirakawa S. Curry C.L. Sage D.R. Libermann T. Dezube B.J. Fingeroth J.D. Detmar M. Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.Nat. Genet. 2004; 36: 683-685Crossref PubMed Scopus (303) Google Scholar, Lagunoff et al., 2002Lagunoff M. Bechtel J. Venetsanakos E. Roy A.M. Abbey N. Herndier B. McMahon M. Ganem D. De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells.J. Virol. 2002; 76: 2440-2448Crossref PubMed Scopus (165) Google Scholar, Moses et al., 1999Moses A.V. Fish K.N. Ruhl R. Smith P.P. Strussenberg J.G. Zhu L. Chandran B. Nelson J.A. Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8.J. Virol. 1999; 73: 6892-6902Crossref PubMed Google Scholar, Naranatt et al., 2004Naranatt P.P. Krishnan H.H. Svojanovsky S.R. Bloomer C. Mathur S. Chandran B. Host gene induction and transcriptional reprogramming in Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)-infected endothelial, fibroblast, and B cells: insights into modulation events early during infection.Cancer Res. 2004; 64: 72-84Crossref PubMed Scopus (140) Google Scholar, Wang et al., 2004Wang H.W. Trotter M.W. Lagos D. Bourboulia D. Henderson S. Makinen T. Elliman S. Flanagan A.M. Alitalo K. Boshoff C. Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma.Nat. Genet. 2004; 36: 687-693Crossref PubMed Scopus (380) Google Scholar). In contrast to the oncogenicity and tumorigenicity of its viral genome, KSHV infection of ECs leads to the induction of KS markers, a spindle cell phenotype, and signs of transformation, but it does not result in the acute angiogenicity and tumorigenicity characteristic of KSHV-infected spindle cells in lesions. This suggests that key oncogenic events are not recapitulated in the KSHV-infected EC cultures. Plausible explanations for this are the tendency for either latency or productivity of the in vitro infections, the possibility that terminally differentiated ECs may not be suitable KS progenitors, or that in vivo growth conditions not mimicked in culture are essential for KSHV oncogenicity. Here, we report that bacterial artificial chromosome (KSHVBac36) transfection of normal mouse bone marrow endothelial-lineage cells induces an angiogenic phenotype and KS-like, KSHV-dependent tumorigenicity. This identifies a cell population containing putative KS progenitors and establishes a KS model with the following characteristics: (1) the pathological phenotype is a consequence of KSHV gene expression in normal progenitor cells subjected to in vivo growth conditions, (2) the histopathologic phenotype of the tumors resembles KS lesions, and (3) the model is suitable for genetic analysis of viral pathogenesis. To create a mouse model of KS, we used as targets of KSHV infection mouse bone marrow adherent-cell preparations enriched in endothelial-lineage cells (mECs) containing mature ECs, their progenitors, and proangiogenic hematopoietic cells (Boshoff and Weiss, 2002Boshoff C. Weiss R. AIDS-related malignancies.Nat. Rev. Cancer. 2002; 2: 373-382Crossref PubMed Scopus (297) Google Scholar, Rafii and Lyden, 2003Rafii S. Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nat. Med. 2003; 9: 702-712Crossref PubMed Scopus (1410) Google Scholar). To be able to select and track infected cells, we transfected mECs with KSHVBac36, which encodes the full infectious KSHV genome in a hygromycin (Hyg)-resistant and EGFP-encoding bacterial artificial chromosome (KSHVBac36) (Zhou et al., 2002Zhou F.C. Zhang Y.J. Deng J.H. Wang X.P. Pan H.Y. Hettler E. Gao S.J. Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.J. Virol. 2002; 76: 6185-6196Crossref PubMed Scopus (209) Google Scholar). mECs transfected with the Bac backbone vector (mEC-V) were used as control. After 3 weeks of Hyg selection, both KSHVBac36-transfected mECs (mECK36) and mEC-V formed contact-inhibited, stromal cell-like monolayers. We found that mECK36 cultures were over 95% EGFP+ (Figure 1A), indicating the presence of KSHVBac36. To determine if the EGFP+ cell population was infected by KSHV, we analyzed the expression of the latent genes LANA and Kaposin by immunofluorescence. As shown in Figures 1A and 1B, all EGFP+ mECK36 were positive for LANA and displayed the classic nuclear punctuated pattern produced by the tethering of KSHV episomes to the host chromosome (Ballestas et al., 1999Ballestas M.E. Chatis P.A. Kaye K.M. Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen.Science. 1999; 284: 641-644Crossref PubMed Scopus (586) Google Scholar). As shown in Figure 1C, mECK36 also stained for the latent oncogene Kaposin (Muralidhar et al., 1998Muralidhar S. Pumfery A.M. Hassani M. Sadaie M.R. Azumi N. Kishishita M. Brady J.N. Doniger J. Medveczky P. Rosenthal L.J. Identification of kaposin (open reading frame K12) as a human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) transforming gene.J. Virol. 1998; 72: 4980-4988Crossref PubMed Google Scholar) and displayed its characteristic perinuclear localization. To estimate the integrity of the KSHV genome in mECK36, we performed real-time RT-PCR analysis for the entire KSHV transcriptome of mECK36 (Dittmer, 2003Dittmer D.P. Transcription profile of Kaposi's sarcoma-associated herpesvirus in primary Kaposi's sarcoma lesions as determined by real-time PCR arrays.Cancer Res. 2003; 63: 2010-2015PubMed Google Scholar, Fakhari and Dittmer, 2002Fakhari F.D. Dittmer D.P. Charting latency transcripts in Kaposi's sarcoma-associated herpesvirus by whole-genome real-time quantitative PCR.J. Virol. 2002; 76: 6213-6223Crossref PubMed Scopus (178) Google Scholar). We compared the expression pattern in the endothelial-lineage (mECK36) cells with non-KSHV targets (Bac36-transfected NIH 3T3 cells) (C.C., A.D.M., and E.A.M., unpublished data). mRNA levels were 218-fold higher (CV: 45…391, n = 100, p ≤ 10−14) in mECK36 than in Bac36-transfected NIH 3T3 cells. In contrast, KSHV latent mRNAs (LANA, lat273f, orf72, Taq-F4, 73-5′UTR, orf72f1) were present at about equal levels, with a mean difference of 2.24-fold (CV: −1.04.5.51, n = 6, p ≤ 0.29). This shows that mECK36 have increased lytic gene expression. All KSHV genes are expressed over background levels (Figure 1F), indicating that the KSHV transcriptome is complete in mECK36, further suggesting that the Bac has not undergone major deletions or translocations that would affect KSHV gene expression. Although lytic gene transcription in mECK36 is abundant, they did not produce KSHV virions, as indicated by transmission electron microscopy analysis and the absence of a cytopathic effect (CPE) (data not shown). Double immunofluorescence staining for the latent antigen LANA and the late lytic antigen K8.1 shows a coexpression pattern (Figures 1D and 1E) in most of the cells, which, together with the mECK36 KSHV transcription profile, is indicative of an abortive lytic transcription status. The abortive lytic status of mECK36 is expected to be highly oncogenic, since it combines the expression of latent and lytic KSHV genes with transforming and angiogenesis-inducing potential. Both mEC-V and mECK36 became immortalized in culture and could be passaged infinitely (current passages > 50). Since murine cells express telomerase, they generally become immortalized as a consequence of cell culture shock (Sherr and DePinho, 2000Sherr C.J. DePinho R.A. Cellular senescence: mitotic clock or culture shock?.Cell. 2000; 102: 407-410Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). KSHV genes can upregulate telomerase expression (Knight et al., 2001Knight J.S. Cotter 2nd, M.A. Robertson E.S. The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus transactivates the telomerase reverse transcriptase promoter.J. Biol. Chem. 2001; 276: 22971-22978Crossref PubMed Scopus (95) Google Scholar); therefore, we tested telomerase activity in mEC-V and mECK36 and found increased levels in mECK36 (Figure 2A). Transformed cells have increased telomerase expression; yet, the finding that mECK36 were contact inhibited suggested that they were not transformed. To identify transformed clones among the mECK36 population, we carried out an anchorage-independent growth assay in soft agar by using NIH 3T3 and RasV12-transformed NIH 3T3 cells as negative and positive controls, respectively. While 92% of the RasV12-transformed NIH 3T3 cells formed colonies in soft agar, neither mECK36, mEC-V, nor control NIH 3T3 cells formed colonies, suggesting that mECK36 are not transformed. To investigate KSHVBac36-induced angiogenicity, we determined VEGF secretion levels, mRNA expression of angiogenic markers, and skin angiogenicity in mEC-V and mECK36. As shown in Figures 2B–2E, KSHVBac36 transfection of mECs increased VEGF secretion levels, upregulated ligands and receptors of the VEGF and Angiopoietin family, and displayed a significantly increased ability (p < 0.05) to induce microvessel formation in mice skin. Taken together, these results indicate that KSHV expression in mECs induces an angiogenic phenotype. Cell angiogenicity is key for tumor growth. To determine if mECK36 were tumorigenic in mice, they were injected subcutaneously in nude mice by using mEC-V as controls. We found that mECK36 formed solid tumors 3 weeks after injection, in contrast to mEC-V, which did not (Figures 3A–3C). Upon dissection, we found that the tumors were green-yellow, indicating a significant percentage of EGFP+ cells (Figure 3B). Tumors were analyzed in a blind fashion and were found to be “vascularized spindle cell sarcomas,” which is the histological presentation of human KS tumors (Figure 3D). Advanced invasive KS often appears as multifocal lesions on the lungs and the gastrointestinal tract (Safai et al., 1985Safai B. Johnson K.G. Myskowski P.L. Koziner B. Yang S.Y. Winningham-Ruddles S. Godbold J.H. Dupont B. The natural history of Kaposi's sarcoma in Acquired Immunodeficiency Syndrome.Ann. Intern. Med. 1985; 103: 744-750Crossref PubMed Scopus (209) Google Scholar). To determine whether mECK36 could also induce invasive KS, we injected passage 30 cells intravenously in irradiated SCID/NOD mice. A total of 3 months after injection, mECK36-injected animals were sick, while mEC-V-injected animals did not show any signs of ailment. Necropsy of mECK36-injected animals revealed multifocal, invasive spindle cell sarcoma lesions in lungs (9–20 foci per lung) (Figures 3E and 3F), a presentation reminiscent of advanced visceral KS. The fact that subcutaneous tumors showed strong EGFP fluorescence (Figure 4A) indicated the presence of mECK36 in the tumors. Although mECK36 did not display typical signs of cell transformation in vitro, they were malignant in vivo, forming vascularized tumors that led to significant morbidity and mortality. To determine the presence of KSHV in the EGFP+ spindle cells of mECK36 tumors, we carried out immunohistochemical determination of KSHV LANA expression. Figures 4B and 4C show that LANA+ cells comprise 30%–50% of mECK36 tumors, which is also reminiscent of KS lesions (Dupin et al., 1999Dupin N. Fisher C. Kellam P. Ariad S. Tulliez M. Franck N. van Marck E. Salmon D. Gorin I. Escande J.P. et al.Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.Proc. Natl. Acad. Sci. USA. 1999; 96: 4546-4551Crossref PubMed Scopus (595) Google Scholar). To further characterize the phenotype of the KSHV-harboring cells in tumors and the status of their KSHV infection, we costained for KSHV LANA, podoplanin, and K8.1. All EGFP+ cells coexpressed LANA and podoplanin (Figure 4B). Confocal images at different Z planes revealed that up to 90% of nuclei were LANA+ and displayed the punctuated pattern indicative of episomal infection (Figure 4D). More than 20% of the tumor cells were also K8.1+ (Figure 4E). To determine changes in the status of KSHV infection in mECK36 due to in vivo growth conditions, we compared the KSHV transcription profiles from three different tumors and from mECK36 in culture (Figure 4F) normalized for LANA in order to include only KSHV-harboring cells (Dittmer, 2003Dittmer D.P. Transcription profile of Kaposi's sarcoma-associated herpesvirus in primary Kaposi's sarcoma lesions as determined by real-time PCR arrays.Cancer Res. 2003; 63: 2010-2015PubMed Google Scholar). Compared to cultured mECK36, the tumors showed a 15.6-fold increased expression of lytic genes versus a 1.1-fold increase of latent genes. Since the average variation among the three tumors was only 2.13-fold (CV: 1.93.2.33, n = 91), the increase in lytic mRNA levels must represent a biological response of the viral genome to in vivo growth conditions, rather than a variation introduced by intertumor variation. Despite increased lytic transcription, mECK36 did not show increased viral genome replication, evidenced by the lack of variations in the KSHV viral load and the absence of KSHV virions in the tumors (data not shown). These data indicate that, similar to mECK36 in culture, mECK36 tumors express latent and lytic genes, consistent with an abortive lytic infection, but display increased expression of KSHV lytic genes. To further determine if the phenotypic markers of the mECK36 sarcomas corresponded to those typically found in human KS lesions, we immunostained for CD31/PECAM (pan-EC marker), VEGF-R2 (EPCs, angiogenically active vessels, KS), and CD34 (EPC, microvascular endothelium, and KS) (Boshoff and Weiss, 2002Boshoff C. Weiss R. AIDS-related malignancies.Nat. Rev. Cancer. 2002; 2: 373-382Crossref PubMed Scopus (297) Google Scholar, Brown et al., 1996Brown L.F. Tognazzi K. Dvorak H.F. Harrist T.J. Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.Am. J. Pathol. 1996; 148: 1065-1074PubMed Google Scholar, Rafii and Lyden, 2003Rafii S. Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration.Nat. Med. 2003; 9: 702-712Crossref PubMed Scopus (1410) Google Scholar). The presentation of these markers (Figure 5A) corresponded to a highly vascularized tumor (CD31+ vessels) bearing some VEGF-R2+ vessels as well as VEGF-R2+ and CD34+ cells. To assess the expression of VEGF receptors in EGFP+ cells, we used immunofluorescence and cytofluorometry analysis for the VEGF-R1 (ECs, proangiogenic hematopoietic cells), VEGF-R2, and VEGF-R3 (lymphatic endothelium and KS) (Hong et al., 2004Hong Y.K. Foreman K. Shin J.W. Hirakawa S. Curry C.L. Sage D.R. Libermann T. Dezube B.J. Fingeroth J.D. Detmar M. Lymphatic reprogramming of blood vascular endothelium by Kaposi sarcoma-associated herpesvirus.Nat. Genet. 2004; 36: 683-685C" @default.
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• W1978855492 title "In Vivo-Restricted and Reversible Malignancy Induced by Human Herpesvirus-8 KSHV: A Cell and Animal Model of Virally Induced Kaposi's Sarcoma" @default.
• W1978855492 cites W100681333 @default.
• W1978855492 cites W101219436 @default.
• W1978855492 cites W134197049 @default.
• W1978855492 cites W1521064197 @default.
• W1978855492 cites W1523189699 @default.
• W1978855492 cites W1567318029 @default.
• W1978855492 cites W1578101184 @default.
• W1978855492 cites W1972167991 @default.
• W1978855492 cites W1977500292 @default.
• W1978855492 cites W1979103623 @default.
• W1978855492 cites W1979733640 @default.
• W1978855492 cites W1982047106 @default.
• W1978855492 cites W1987162010 @default.
• W1978855492 cites W1999762190 @default.
• W1978855492 cites W2000000541 @default.
• W1978855492 cites W2002155325 @default.
• W1978855492 cites W2009714338 @default.
• W1978855492 cites W2010558751 @default.
• W1978855492 cites W2019745410 @default.
• W1978855492 cites W2024374396 @default.
• W1978855492 cites W2032438721 @default.
• W1978855492 cites W2044627970 @default.
• W1978855492 cites W2046245571 @default.
• W1978855492 cites W2051260092 @default.
• W1978855492 cites W2061789335 @default.
• W1978855492 cites W2065537276 @default.
• W1978855492 cites W2066410692 @default.
• W1978855492 cites W2066418230 @default.
• W1978855492 cites W2069162926 @default.
• W1978855492 cites W2073648680 @default.
• W1978855492 cites W2079656132 @default.
• W1978855492 cites W2084391519 @default.
• W1978855492 cites W2086517030 @default.
• W1978855492 cites W2089097621 @default.
• W1978855492 cites W2089470376 @default.
• W1978855492 cites W2091859075 @default.
• W1978855492 cites W2102030590 @default.
• W1978855492 cites W2102323376 @default.
• W1978855492 cites W2104235597 @default.
• W1978855492 cites W2108819945 @default.
• W1978855492 cites W2109745177 @default.
• W1978855492 cites W2113694619 @default.
• W1978855492 cites W2120246830 @default.
• W1978855492 cites W2121964400 @default.
• W1978855492 cites W2125325745 @default.
• W1978855492 cites W2130399704 @default.
• W1978855492 cites W2136357286 @default.
• W1978855492 cites W2136620152 @default.
• W1978855492 cites W2143090686 @default.
• W1978855492 cites W2144126736 @default.
• W1978855492 cites W2145561586 @default.
• W1978855492 cites W2149453948 @default.
• W1978855492 cites W2154331370 @default.
• W1978855492 cites W2154666567 @default.
• W1978855492 cites W2156618078 @default.
• W1978855492 cites W2160768318 @default.
• W1978855492 cites W2166187451 @default.
• W1978855492 cites W2168492746 @default.
• W1978855492 cites W2170125822 @default.
• W1978855492 cites W2324248025 @default.
• W1978855492 cites W2325680067 @default.
• W1978855492 cites W2336241188 @default.
• W1978855492 cites W2339279771 @default.
• W1978855492 cites W2409900806 @default.
• W1978855492 cites W253295397 @default.
• W1978855492 cites W3112188057 @default.
• W1978855492 cites W4254613993 @default.
• W1978855492 cites W72344367 @default.
• W1978855492 doi "https://doi.org/10.1016/j.ccr.2007.01.015" @default.
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