Protection of murine B cells from apoptosis by a combination of IL4, PMA, dextran sulfate and low dose cycloheximide

Murine B lymphocytes undergo apoptosis when cultured in vitro, with 40-50% losing their membrane phospholipid asymmetry as measured by merocyanine staining (MC540), and 30-40% becoming hypodiploid by 16 h of culture. Phorbol myristate acetate (PMA) (10 ng/ml), IL4 (25U/ml), low dose cycloheximide (0.1ug/ml), and dextran sulfate (DXS) (5ug/ml) all reduced apoptosis when used individually, but in combination (COMBO) they profoundly inhibited apoptosis, to less than 2% MC540+ cells at 16 h. COMBO strongly induced bcl-xL mRNA and c-myc mRNA by 4 h. c-Myc protein was increased by 4 h, and was decreasing by 16 h., while Bcl-xL protein was detectable by 4 h and was still increasing by 16 h. Combinations which included DXS appeared to decrease levels of Bax protein. COMBO cultures exhibited extensive homotypic adhesion, which depended on the presence of both DXS and PMA. COMBO treated cells analyzed as late as 72 h of culture were approximately 80% in Go, with less than 5% apoptotic, and less than 10% in S phase or G2/M. COMBO treated cells could be re-stimulated by treatment with LPS. We conclude that increased Bcl-xL and c-Myc expression, in conjunction with reduced expression of Bax correlate with prolonged survival of murine B cells in vitro.