GERMINAL CENTER-DERIVED SIGNALS ACT WITH BCL-2 TO DECREASE APOPTOSIS AND INCREASE CLONOGENICITY OF DRUG-TREATED HUMAN-B-LYMPHOMA-CELLS

Bcl-2 suppresses drug-induced apoptosis in vitro, although in many cas es, this results only in a delayed onset of cell death, In vivo surviv al signals from the extracellular environment may also contribute to d rug resistance and may act with Bcl-2 to promote long-term cell surviv al, Ligation of CD40 on B-lymphocytes in germinal centers (GCs) can su ppress apoptosis induced by calcium ionophore or anti-IgM in vitro, We asked whether a combination of Bcl-2 expression and the provision of a culture environment that mimicked that of the GC [CD40 ligation and interleukin 4 (IL-4)] could increase the ability of B lymphoma cells t o resist drug-induced apoptosis, A Burkitt lymphoma (BL) cell line tra nsfected with either human bcl-2 (BL-bcl-2) or control plasmid (BL-Sv2 ) was used to examine the effects of Bcl-2 overexpression on the cellu lar response and long-term survival after treatment with the DNA-alkyl ating drug chlorambucil (CMB) in the presence or absence of CD40 ligat ion and IL-4, Administration of 20 mu m CMB completely prevented cell proliferation, This was associated with an increase in p53 protein lev els within 24 h, without an elevation in p21, Bax, or Mdm2 proteins. A nalyses of cell cycle distribution and of cyclin B expression demonstr ated that both cell lines arrested at G(2)/M, where they died, Fifty % of BL-Sv2 cells died within 2 days, whereas 50% cell death was not ob served in the BL-bcl-2 cultures until 6 days had passed, Cross-linking of CD40 with a monoclonal antibody elevated Bcl-x(L) protein levels b y 3 h and also provided a delay in CMB-induced death, Ninety-six h aft er the addition of 20 mu M CMB, 78% of the BL-Sv2 cells were apoptotic , whereas ligation of CD40 on BL-Sv2 cells reduced the proportion of a poptotic cells to 38%, Overexpression of Bcl-2 (in BL-bcl-2 cells) red uced apoptosis to 41%, However, when the BL-bcl-2 cells were treated w ith CMB together with ligation of CD40, apoptosis was reduced further to only 17% at 96 h, The Bcl-2-mediated delay in the execution of CMB- induced apoptosis did not translate significantly to increased clonoge nicity, In contrast, the provision of BL-Sv2 cells with an ability to interact with the adhesion molecule vascular cell adhesion molecule-1, CD40 ligation, and IL-4 significantly increased clonogenic survival, and this,vas improved in BL-bcl-2, cells exposed to these GC-derived s ignals, These data demonstrate that the kinetics of drug-induced apopt osis can he modulated by Bcl-2 as well as by IL-4, vascular cell adhes ion molecule-1, and CD40 ligation, the latter possibly involving the f unction of Bcl-x(L). That these factors appear to act together to enha nce proliferative potential after DNA damage has important implication s regarding the development of drug resistance in B-cell lymphomas and future strategies for improved chemotherapy.