IntroductionAngiogenesis has a critical role in the pathophysiology and progression of multiple myeloma (MM) supporting the growth and survival of MM cells. [1][2][3][4][5] The angiogenic process in MM is sustained mainly by the overexpression of proangiogenic factors directly by MM cells including VEGF, 6 angiopoietin-1 (ANG-1), 7 osteopontin (OPN), . 9 Nevertheless, the molecular mechanisms underlying the regulation of angiogenesis in MM have not been completely elucidated.The new candidate tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of tumor growth and angiogenesis through the association with NF-B. ING4 is a nuclear factor expressed in all normal tissues and markedly reduced in glioblastoma cells and head and neck squamous cell carcinoma, with levels inversely correlated with tumor grade. 10,11 Inhibition of ING4 expression strongly promotes the growth of glioma cells in vivo, whereas its overexpression leads to growth inhibition through ING4's capability to interact with p65 subunit of NF-B. 10 Interestingly, it has been also shown that tumors lacking ING4 showed increased vascularization compared with ING4-expressing tumors. 12 Moreover ING4 down-regulated the angiogenic-related molecules including IL-8 and the hypoxia inducible factor-1␣ (HIF-1 ␣) activity in hypoxic condition through the involvement of HIF prolyl hydroxylase 2 (HPH-2) 10,13 In turn, the role of hypoxia has been recently highlighted in the promotion of angiogenesis. 14 The expression of ING4 by MM cells, as well as its potential role in MM-induced angiogenesis, has never been investigated. In this study, we evaluated the expression of ING4 in malignant MM cells and the potential relationship between ING4 and the production of proangiogenic molecules by MM cells in normoxic and hypoxic conditions, as well as its relationship with the "in vitro and in vivo" angiogenesis. Submitted February 15, 2007; accepted September 4, 2007. Prepublished online as Blood First Edition paper, September 11, 2007; DOI 10.1182 DOI 10. /blood-2007 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on August 29, 2018. by guest www.bloodjournal.org From
Patients, materials, and methods
Cells and cell culture conditionsCell lines. Human myeloma cell lines (HMCLs) XG-6, XG-1, and JJN3 were obtained from Dr Bataille (Nantes, France). U266 was obtained from the American Type Culture Collection (Rockville, MD). OPM2 and RPMI-8226 were purchased from DSM (Braunschweig, Germany). ARP-1 and H929 were generously received from Dr Shaughnessy's laboratory (Little Rock, AR).Cell cultures. HMCLs were incubated in RPMI medium at 10% FCS (Invitrogen Life Technologies, Milan, Italy) and maintained with or without IL-6 (3 ng/mL; Endogen Woburn, MA). In a series of experiments, HMCLs were incubated with the HPH-2 in...
