Abstract-This study was designed to identify cellular responses associated with free cholesterol (FC) accumulation in model macrophage foam cells. Mouse peritoneal macrophages (MPMs) or J774 macrophages were loaded with cholesteryl esters using acetylated LDL and FC/phospholipid dispersions and were subsequently exposed to an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. This treatment produced a rapid accumulation of cellular FC. The FC that accumulated due to ACAT inhibition was more readily available for efflux to 2-hydroxypropyl-␤-cyclodextrin (which removes cholesterol from the plasma membrane) than FC in untreated control cells. After a 3-hour exposure to an ACAT inhibitor, a significant increase in phospholipid synthesis was seen, followed by the leakage of LDH after 12 hours of treatment.

The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-1 (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34cdc2 kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34CDK2/cyclin E and p34CDK2/cyclin A, but not for p34CDK4/cyclin D1 or p34cdc2/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-1 (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67% and 90% respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.

Accessory cells are required for the induction of lymphocyte proliferation in response to mitogens or antigens. Rat pulmonary cells were tested for the presence of accessory activity in lymphocyte proliferation induced by sodium periodate. Buffer-perfused lungs were excised, minced, and enzymatically digested. The resulting pulmonary cells (PC) were separated into high density (HD-PC, 32 to 57%) and low density (LD-PC, 9 to 32%) fractions in a discontinuous density gradient of bovine plasma albumin (BPA). Both macrophages and dendritic cells were observed in the LD-PC by light microscopy. HD-PC, LD-PC, adherent LD-PC, nonadherent LD-PC, and a purified preparation of pulmonary dendritic cells (DC-P) were tested for accessory activity in the presence of periodate-treated, lymph-node-derived lymphocytes as responders. Most of the accessory activity was found in the LD-PC. Increasing numbers of LD-PC stimulated proliferation of responder lymphocytes in a linear, dose-dependent manner; higher numbers had a suppressive effect. Nonadherent LD-PC containing dendritic cells also produced a dose-dependent increase in periodate-induced lymphocyte proliferation, whereas adherent LD-PC, morphologically identified as macrophages, were suppressive. Removal of phagocytic macrophages from nonadherent LD-PC resulted in an eightfold increase in both the percent of DC-P present and the amount of accessory activity in the LD-PC. We conclude that pulmonary dendritic cells are potent accessory cells for periodate-induced lymphocyte proliferation.