Mouse Anti-Sheep Erythrocyte Hybridoma [TSCD-SQ9-29-26]

Epirubicin is a cytotoxic drug used in the treatment of different types of cancer and increasing evidence suggests that its target is cell membranes. In order to gain insight on its toxic effects, intact red blood cells (RBC), human erythrocyte membranes and molecular models were used. The latter consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes found mainly in the outer and inner monolayers of the human erythrocyte membrane, respectively. The results obtained by X-ray diffraction displayed that epirubicin induced structural perturbations in multilayers of DMPC. Differential scanning calorimetry (DSC) showed that epirubicin disturbed the thermotropic behavior of both DMPC and DMPE vesicles, whereas fluorescence spectroscopy demonstrated alterations in the fluidity of DMPC vesicles and the erythrocyte membrane. Scanning electron microscopy (SEM) revealed that epirubicin changed the normal discoid form of RBC to echinocytes and stomatocytes. Electron paramagnetic resonance (EPR) disclosed that this drug induced conformational changes in the erythrocyte membrane proteins. These findings demonstrate that epirubicin interacts with lipids and proteins of the human erythrocyte membrane, effects that might compromise the integrity and function of cell membranes. This is the first time that its toxic effects on the human erythrocyte membrane have been described.

We use phase contrast microscopy of red blood cells to observe the transition between the initial discocyte shape and a spiculated echinocyte form. During the early stages of this change, spicules can move across the surface of the cell; individual spicules can also split apart into pairs. One possible explanation of this behaviour is that the membrane forms large scale domains in association with the spicules. The spicules are formed initially at the rim of the cell and then move at speeds of up to 3 mu m/min towards the centre of the disc. Spicule formation that was reversed and then allowed to proceed a second time resulted in spicules at reproducible places, a shape memory effect that implies that the cytoskeleton contributes towards stopping the spicule movement. The splitting of the spicules produces a well-defined shape change with an increase in membrane curvature associated with formation of the daughter pair of spicules; the total boundary length around the spicules also increases. Following the model in which the spicules are associated with lipid domains, these observations suggest an experimental procedure that could potentially be applied to the calculation of the line tension of lipid domains in living cells.