IntroductionKaposi sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is the latest addition to the list of human herpesviruses. KSHV was first isolated from Kaposi sarcoma (KS) lesions in persons suffering from acquired immunodeficiency syndrome (AIDS) in 1994. 1 The cancerous conditions that are etiologically associated with KSHV are KS, primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). 2 Apart from the inflammatory cytokines (ICs)/growth factors (GFs), lytic KSHV infection plays an instrumental role in the progression of KS lesions. 3 The lytic cycle of infection is also critical for the spread of KSHV to different organs. Successful KSHV infection is characterized by both virus entry and the ability of the virus to establish latency.In our earlier study, we provided evidence to show that the overexpression of Raf specifically enhanced KSHV infection of target cells at the level of entry. 4 Regulation of Raf is crucial for the proper maintenance of cell growth, proliferation, apoptosis, and differentiation. 5 Of the 3 isoforms, B-Raf has gained a lot of focus over the last couple of years due to the detection of B-Raf somatic missense mutations in malignant melanomas (66%), colon cancers (15%), and at lower frequencies in a wide variety of human cancers. [6][7][8] The ability of Raf to regulate vascular endothelial growth factor-A (VEGF-A) has also been demonstrated. 4 In separate studies, we found VEGF-A to enhance KSHV infection of fibroblasts and epithelial cells. 9,10 Interestingly, constitutive activation of the components (Ras/Raf) of the mitogen-activated protein kinase (MAPK) pathway of signaling as well as VEGF expression has been a common feature with KSHV pathogenesis. 11,12 Hence, in this study we attempted to analyze the relationship between the expression of B-Raf and VEGF-A in KSHV-infected hematopoietic cells. We provide evidences for the existence of a Raf-dependent VEGF-A expression in KSHV-infected hematopoietic cells.
Materials and methods
Cell cultureHuman foreskin fibroblasts (HFFs; Clonetics, Walkersville, MD), BCBL-1, BC-1 (American Type Culture Collection [ATCC], Manassas, VA; CRL-2230), BC-2 (ATCC CRL-223), BCP-1 (ATCC CRL-2294), and BJAB cells were used in this study. Target cells were grown in either phenol red-free Dulbecco modified Eagle medium (DMEM) or RPMI medium (Invitrogen, Carlsbad, CA) containing 10% charcoal-stripped fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), L-glutamine, and antibiotics. 13,14 Dermal microvascular endothelial cells (HMVEC-Ds; CC-2543; Clonetics) were propagated in EGM MV-microvascular endothelial cell medium (Clonetics) as per standard protocols. 13 The passage numbers for HFFs and HMVEC-Ds used in this study ranged between 6 and 10, and 5 and 9, respectively. HFF/pBabePuro3, HFF/⌬B-Raf [DD] :ER, and HFF/⌬B-Raf [FF] : ER cells were cultured as per standard protocols. 4,10 -Estradiol (1 M) stimulation of these cells results in the activation of ⌬B-Raf:ER fusion proteins. 4Inhibitors PD98059 w...