Infrared spectroscopic study of bryostatin 1-induced membrane alterations in a B-CLL cell line

Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) indu ces significant alterations in the membranes of WSU-CLL cells (a drug-resis tant B-CLL cell line), changes which may play an important role in the mech anism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine ( 2-CdA). However, it is not clear whether the plasma membranes or the mitoch ondria, or both are involved; nor is it known which of these two targets is more important for regaining the cells former drug sensitivity. For the pr esent study, we treated WSUCLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (I R) spectroscopic and chromatographic analyses. IR spectroscopy revealed a d ecreased glycosylation of both plasma membranes and mitochondria in Bryo 1- treated cells compared to untreated cells. The amount of lipid relative to protein was increased in both types of membranes, but considerably more enh anced in the plasma membrane fraction of the Bryo 1-treated cells than in m itochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid con tent in plasma membranes, whereas the lipids in the mitochondria remained e ssentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphat idylethanolamines doubled and sphingomyelins increased five-fold compared t o the lipid composition in plasma membranes of untreated cells. In addition , the IR spectroscopic analysis provided evidence for an increased plasma m embrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitoc hondria remained essentially unchanged; marker bands indicating mitochondri al DNA decreased upon Bryo 1 treatment. These results suggest that Bryo I i ncreases the sensitivity of WSU-CLL cells to chemotherapeutic agents such a s 2-CdA by action on two cell targets: (1) introduction of significant chan ges in plasma membrane permeability or fluidity through modifications in li pid content and composition as well as by reducing the surface glycosylatio n; (2) introduction of changes in lipid and DNA content of the mitochondria . Small alterations in the lipid composition of the mitochondria may provid e the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.