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

Abstract

Previous studies on intact cells have shown that bryostatin 1 (Bryo 1) induces significant alterations in the membranes of WSU-CLL cells (a drug-resistant B-CLL cell line), changes which may play an important role in the mechanism of reduced drug resistance of B-CLL cells to 2-chlorodeoxyadenosine (2-CdA). However, it is not clear whether the plasma membranes or the mitochondria, 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 present study, we treated WSU-CLL cells with Bryo 1, isolated plasma membranes and mitochondria, and then subjected the purified fractions to infrared (IR) spectroscopic and chromatographic analyses. IR spectroscopy revealed a decreased 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 enhanced in the plasma membrane fraction of the Bryo 1-treated cells than in mitochondria. Quantitative lipid analysis by thin layer chromatography also revealed that Bryo 1 treatment significantly increased the phospholipid content in plasma membranes, whereas the lipids in the mitochondria remained essentially unchanged. Changes in lipid composition were quite dramatic for plasma membranes where phosphatidylcholines were decreased by 50%, phosphatidylethanolamines doubled and sphingomyelins increased five-fold compared to the lipid composition in plasma membranes of untreated cells. In addition, the IR spectroscopic analysis provided evidence for an increased plasma membrane fluidity in Bryo 1-treated cells, whereas the fluidity of the mitochondria remained essentially unchanged; marker bands indicating mitochondrial DNA decreased upon Bryo 1 treatment. These results suggest that Bryo 1 increases the sensitivity of WSU-CLL cells to chemotherapeutic agents such as 2-CdA by action on two cell targets: (1) introduction of significant changes in plasma membrane permeability or fluidity through modifications in lipid content and composition as well as by reducing the surface glycosylation; (2) introduction of changes in lipid and DNA content of the mitochondria. Small alterations in the lipid composition of the mitochondria may provide the conditions for an altered proton gradient and transmembrane potential leading to apoptosis and decreased cell survival.