Mechanism of Pax5 Down-Regulation and Its Function in Primary Effusion Lymphoma

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). PEL develops body cavity effusion without tumor masses especially in immunodeficient patients. The prognosis of PEL is very poor ad there is no standard therapy for PEL. Therefore, the molecular pathogenesis of PEL is needed to be clarified to develop novel therapy. The expressions of Pax5 in PEL patient samples were decreased compared with KSHV/HHV-8-negative B-cell lines in the publically available data set. In this study, we analyzed the mechanism of Pax5 down-regulation and performed the restoration of Pax5 in PEL cells.

Methods: Pax5 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). To study the function of Pax5 in PEL, Pax5 was conditionally expressed using tet-on system in three PEL cell lines. To examine the mechanism of Pax5 reduction in PEL, the bisulfite sequencing was performed to analyze the methylation of the Pax5 promoter region in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used for demethylation of Pax5 promoter. The anti-proliferative activities of restoring Pax5 in PEL cell lines were analyzed by the methylthiotetrazole method. Cell cycle profiles were analyzed using flow cytometry. Microarray analysis was performed to detect Pax5-regulated genes. The master regulator of KSHV/HHV-8 viral replication, ORF50 , was determined by quantitative PCR.

Results: The expression of Pax5 mRNA in PEL cell lines was weaker than in the Burkitt lymphoma cell line, Raji, and normal B-cells derived from peripheral blood mononuclear cells, which express Pax5 mRNA. Although Pax5 promoter region of Raji cells was not methylated, that of PEL cells was heavily methylated. The treatment with 5-Azacytidine induced the demethylation of Pax5 promoter region and restored the expression of Pax5 mRNA. Restoration of Pax5 suppressed cell proliferation via cell cycle arrest. Cell cycle-related genes were down-regulated and several B-cell markers were up-regulated in Pax5-induced PEL cells. Pax5 did not induce ORF50 expression.

Conclusions: Pax5 is silenced in PEL cells via the methylation of Pax5 promoter region. Pax5 regulates cell cycle and causes growth inhibition. Reduction of Pax5 has a critical role in the development of PEL and Pax5 is a tumor suppressor in PEL. The targeting of Pax5 with DNA methyltransferase inhibitor could be a novel therapeutic approach in patients with PEL.