Establishment and characterization of a primary effusion (body cavity- based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus

Abstract

The recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity-based lymphomas (BCBL) or primary effusion lymphomas (PEL). These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient, and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B-cell immunogenotype and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.

Cyclin D1 and retinoblastoma protein expression in Kaposi's sarcoma

“…In late KS lesions there is intense transcription of KSHV-cyclin (7), which indicates that the viral cyclin can coexist with the overexpression of cellular cyclin Dl. This is supported by our finding of cyclin Dl overexpression in the BC3 cell line, which also transcribes KSHV-cyclin (20). Hypothetically, it is possible that a synergistic relation between the viral and the cellular cyclins might ensue in this setting.…”

“…The cyclinpositive cell lines A431 and BC3 yielded identical signals. The absence of extra bands in BC3 indicate that the cyclin Dl antibody is highly specific for the cellular cyclin Dl, since this cell line is infected with KSHV and expresses the virally encoded cyclin (20).…”

“…For Western blot analysis, a purified KSHV-encoded cyclin D homolog-bacterial maltose binding fusion protein was tested with the cellular cyclin DI antibody. Total cellular lysates from A431 (a KSHV-negative cell line cloned from squamous cell carcinoma of the vulva (13)) and BC3 (a KSHV-positive cell line cloned from a primary effusion lymphoma (20)) were used as positive controls for the cellular cyclin DL These proteins (5 to 50 ]ig per lane) were separated by SDS-PAGE and transferred to Protran nitrocellulose membranes (Schleicher & Schnell, Keene, NH). Immunodetection with the anti-cyclin DI antibody was performed at a dilution of 1:400 for 60 min at room temperature.…”

Glycyrrhizic acid alters Kaposi sarcoma–associated herpesvirus latency, triggering p53-mediated apoptosis in transformed B lymphocytes

“…Twelve different human cell types were used. BC-3, BCBL-1, and BCP-1 cells are KSHVpositive, EBV-negative B cells (44)(45)(46). BC-1 and BC-2 cells are KSHV-and EBV-positive B cells (47).…”

Egr-1 regulates RTA transcription through a cooperative involvement of transcriptional regulators

“…Hence, a study depicting the direct mechanism of how Egr-1 regulates the expression of RTA is mandatory. To answer these questions, we used BC-3 and BCBL-1 cells, which maintain the viral genome in a latent state [57,58], for quantifying the amounts of virions produced after Egr-1 depletion. The Egr-1 depleted cells were induced with TPA for 72h followed by quantitation of virions, which showed a significant reduction as compared to the control siRNA transfected cells ( Figure 1A, a for BC-3 and Figure 1A, b for BCBL-1).…”