The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.

The antitumor potency of the mTOR inhibitor rapamycin (sirolimus) is the subject of intense investigations. Primary effusion lymphoma (PEL) appears as an AIDSdefining lymphoma and like Kaposi sarcoma has been linked to Kaposi sarcomaassociated herpesvirus (KSHV). We find that (1) rapamycin is efficacious against PEL in culture and in a murine xenograft model; (2) mTOR, its activator Akt, and its target p70S6 kinase are phosphorylated in PEL; (3) rapamycin inhibits mTOR signaling as determined by S6 phosphorylation; (4) KSHV transcription is unaffected; (5) inhibition of IL-10 signaling correlates with drug sensitivity; and (6) IntroductionOrgan transplant recipients are at an increased risk of Kaposi sarcoma (KS) and certain viral lymphomas because iatrogenic T-cell immunosuppression activates the causative agent of KS the Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8). 1-6 KSHV was originally identified in the context of HIV-1-induced T-cell depletion 7 and is, by an overwhelming body of evidence, associated with KS as well as the B-cell lymphoproliferative diseases multicentric Castleman disease and primary effusion lymphoma (PEL). [8][9][10] KHSV normally resides latent in a yet to be defined B-lymphocyte compartment. 11 Immunosuppression is thought to disturb host surveillance of this virus, leading to reactivation, increased systemic viral load, [12][13][14][15] and infection of endothelial cells. KSHV-infected LYVE-1 ϩ (lymphatic vessel hyaluronan receptor-1), CD34 ϩ lymphatic endothelial cells constitute the proliferating tumor cells in a KS lesion. [16][17][18][19][20] In 2005, Stallone et al 21 reported that switching from the immunosuppressant drug cyclosporine A to the immunosuppressant drug rapamycin (sirolimus) cured cutaneous KS in a group of 15 kidney transplant recipients. Over a 3-month period all KS lesions disappeared, whereas graft function remained level. This study separated the immunosuppressive function from the anticancer effect of rapamycin in a clinical setting and prompted us to evaluate rapamycin for PEL.The mammalian target of rapamycin (mTOR) executes essential functions of Akt, which is also called protein kinase B, with regard to cancer cell growth and proliferation (reviewed in Hay 22 ). Akt is among the most frequently activated kinases in human cancer. Receptor-tyrosine kinases activate Akt through the generation of phosphoinositol-3,4,5 phosphate (PI3K), which leads to Akt phosphorylation at Thr 308 (through PI3K-dependent kinase 1) and Ser 473 (through PI3K-dependent kinase 2). Generation of PI3K is counteracted by the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene. Phosphorylation at both Thr 308 and Ser 473 is required for full activation of Akt. Akt phosphorylates and thereby inhibits tuberous sclerosis complex 2 (TSC2), which heterodimerizes with TSC1. The TSC1-TSC2 heterodimer has GTPase activity, which inhibits the small G protein Rheb. Rheb is required for mTOR activation. Hence, TSC1 and TSC2 are considered tumor suppressor ...

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is believed to be the causative agent for Kaposi's sarcoma (KS) (for a review, see references 1 and 27). Within KS tumor lesions, the majority of cells express endothelial markers and are latently infected with KSHV, as defined by the presence of the circular viral genome and limited viral-gene expression. Studying KSHV's role in KS is complicated by the fact that cells explanted from KS lesions lose the KSHV genome after several cell divisions in culture (2, 43). In addition to KS, KSHV is associated with two lymphoproliferative diseases: primary effusion lymphomas (PEL) and multicentric Castleman's disease (MCD) (11, 59). In contrast to KS lesions, PEL-derived cell lines that are latently infected with KSHV are readily established in culture. These cells maintain viral episomes indefinitely and remain dependent on KSHV for survival (31,33). Therefore, many aspects of KSHV biology have been studied in PEL-derived cell lines rather than in endothelial cells (56; for a review, see reference 1).Several endothelial-cell-derived tissue culture models have been described. Common to these models, which are based on dermal microvascular endothelial cells (DMVEC), are their susceptibility to cell-free infection with PEL-derived KSHV and their capabili...

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3V-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size. (Cancer Res 2006; 66(7): 3658-66)

Kaposi sarcoma-associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA's contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM + IgD + B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.