Leukemia (2001) 15, 440–444  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Reversible erythrocyte skeleton destabilization is modulated by beta-spectrin phosphorylation in childhood leukemia S Perrotta 1 , E Miraglia del Giudice 1 , A Iolascon 2 , M De Vivo 1 , D Di Pinto 1 , S Cutillo 1 and B Nobili 1 1 Dipartimento di Pediatria, II Universita ` di Napoli, Napoli; and 2 Dipartimento di Biomedicina dell’Eta ` Evolutiva, Universita ` di Bari, Bari, Italy The erythrocyte skeleton plays an essential role in determining the shape and deformability of the red cell. Disruption of the interaction between components of the red cell membrane skel- eton may cause loss of structural and functional integrity of the membrane. Several observations based on studies in vitro strongly suggest that phosphorylation may modify interactions between proteins, leading to a reduced affinity. In particular, increased phosphorylation of -spectrin decreases membrane mechanical stability. In order to investigate the presence of membrane protein defects we investigated the erythrocyte membrane protein composition and phosphorylation in 22 chil- dren with leukemia at diagnosis and during the remission phase. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML). Ten patients (eight ALL and two CML) displayed elliptocytosis and poikilocytosis, an increase of spectrin dimers (41.8 ± 15.6) and an enhanced phosphoryl- ation of -spectrin (108 ± 15%) at diagnosis. These alterations disappeared during the remission phase. This is the first dem- onstration of a reversible erythrocyte membrane alteration in leukemia. Since the -spectrin phosphate sites are located near the C-terminal region and close to the head of the -chain that is involved in dimer-dimer interaction, we supposed that the - chain phosphorylation has an effect upon the interactions between spectrin dimers, ie the tetramerization process. The weakening of this process should be responsible for the pres- ence of elliptocytes and poikilocytes as reported in hereditary elliptocytosis and pyropoikilocytosis. Leukemia (2001) 15, 440–444. Keywords: childhood leukemia; spectrin; phosphorylation; cytoske- leton; elliptocytosis Introduction The red blood cell membrane is one of the major determinants of the ability of erythrocytes to cross repeatedly through the microcirculation. 1 The skeleton that laminates the inner side of the red cell membrane plays an essential role in determin- ing the shape and deformability of the red cell. 1 Defects and deficiencies in the erythrocyte membrane protein structure have been described in several hereditary and acquired anae- mias, where they give rise to mechanically and/or thermally unstable erythrocytes with shortened life spans. 2–4 Spectrin, the most abundant skeletal protein, consists of two chains,  and , intertwined in an antiparallel manner to form dimers. 5 Self-association of spectrin dimers into tetramers is perhaps the best-characterised interaction of membrane proteins, allowing the erythrocyte to acquire its mechanical properties. 6 Disruption of spectrin self-association has been shown to lead to hereditary elliptocytosis and pyropoikilocytosis. 2,3,6,7 Defects of the COOH-terminal end of the -chain, ie the ‘head’ of the molecule, may lead to impaired spectrin self- association. 6 -spectrin consists of 17 homologous 106-amino Correspondence: S Perrotta, Dipartimento di Pediatria, II Universita ` di Napoli, Via S Andrea delle Dame, 4, 80138 Napoli, Italy; Fax: + 390815665403 Received 14 September 2000; accepted 24 October 2000 acid repeat segments and a short nonhomologous COOH-ter- minal segment that contains a consensus sequence for at least four casein kinase I phosphorylation sites. 8 Evidence shows that the shape and the deformability of the red blood cell membrane are very sensitive to the state of phosphorylation. 9–18 In particular, increased phosphorylation of -spectrin decreases membrane mechanical stability. 19 Several acquired abnormalities of erythrocytes and platelets have been described in leukemia and myelodysplastic syn- dromes. Some examples of these abnormalities are acquired thalassaemia, 20,21 abnormal piruvate kinase, 22 size and shape changes of erythrocytes, 23–25 erythrocyte membrane defects, 26–33 platelet defects of von Willebrand factor 34 and of membrane glycoproteins. 35 Here, we further investigated the erythrocyte membrane abnormalities in childhood leukemias. Our studies demon- strate that an erythrocyte skeletal destabilization is due to an increase of the -spectrin phosphorylation and that it disappears during the remission phase. Materials and methods Subjects All blood samples were obtained from 22 patients (age 9.5 ± 2.7 years) and healthy volunteers after informed consent. Sixteen children had acute lymphoblastic leukemia (ALL), three had chronic myeloid leukemia (CML) and three had acute myeloid leukemia (AML) (Table 1). None of the patients had been on any pharmacological treatment for at least 4 weeks prior to the study. Haemoglobin level, reticulocyte count, peripheral blood smear and RBC osmotic fragility was evaluated at diagnosis and during the remission phase. Erythrocyte membrane protein analysis The methods used to analyze the erythrocyte membrane pro- teins have previously been described 36,37 and included: (1) erythrocyte membrane preparation; (2) analysis of the red cell membrane proteins by sodium dodecyl sulphate-polyacrylam- ide gel electrophoresis (SDS-PAGE) (3.5–17% exponential gradient Fairbanks gels and 4–12% linear Laemmli gels); (3) spectrin extraction at 4°C and relative determination of spec- trin dimers and spectrin tetramers by non-denaturing gel electrophoresis (PAGE) at 4°C; and (4) limited tryptic digestion of spectrin. Biochemical analysis was performed at diagnosis and during the remission phase. Phosphorylation of intact cells Red cells from controls and patients were washed in the incu- bation buffer: sodium chloride, 120 mm; sodium bicarbonate,