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The role of proteolysis and phosphorylation of cytoskeletal proteins in human erythrocytes discocyte-echinocyte transition and the effect of adriamycin
Cell Biology
International
Reports,
Vol. 11, No. 3, March
The role proteins in the effect K.Y. Tang, Chemotherapy University,
of proteolysis and phosphorylation human erythrocytes discocyte-echinocyte of Rdriamycin. M.G. Thompson and J.A. Hickman, Pharmaceutical Sciences Group, Birmingham B4 7ET, U.K.
1987
of
249
cytoskeletal transition and
CRC Experimental Institute, Aston
The anthracycline antibiotic, Adrianlycin (ADR), is believed to exert its antitumour effect by intercalation of DNA. However, there is increasing evidence that ADR may exert its toxicity via In the present study, X interactions with the plasma membrane. human erythrocyte was used as a model to investigate the effect of Under various condiADR on membrane-cytoskeleton interactions. tions, human erythrocytes undergo.morphological transitions, which are believed to be regulated by the*+membrane and cytoskeleto in the presence of a Cahi llhen erythrocytes were treated with Ca they underwent concentration-dependent and ionophore (A231871, In the presence time-dependent discocyte-echinocyte transitions. of 1 mM calcium, the morphological transition to echinocytes could be observed as early as 30 seconds after the addition of 5 M SDS-PAGE analysis of the isolated membranes from Ca2+A23187. revealed the breakdown of two major treated erythrocytes cytoskeletal proteins: band 2.1 (ankyrin Mr 200,000) and band 4.1 (Mr 82,000). The breakdown of band 2.1 was indicated by the increase in band 2.3 (Mr 174,000) which is believed to be the proteolytic product of band 2.1. The increase in band 2.3 occurred in parallel with the morphological transition. The breakdown of band 4.1 was only observed after 30 mins treatment with 1 mM calcium. ADi)+was found capable of exerting a protective echinocytosis of human erythr effect agaiF?t the Ca -induced cytes at Ca concentrations of 0 to 150 ,M. Treatment with Ca8+ (150 pM) in the presence of the ionophore A23187 (5 ,JM), produced approximately 70-80% echinocytes after 5 mins. In contrast, there were only 15-20% echinocytes when erytp+ocytes were pre-incubated with ADR (10 MM) for 10 mins. At Ca concentrations of above 200 pM, pre-incubation with ADR had no significant effect on the formation of echinocytes. SDS-PAGE analysis indicated ADR had no significant effect on calcium-mediated cross-linking of cytoskeletal prote@s but inhibited the calcium-mediated breakdown of ankyrin at Ca concentrations of up to 400 u M. Thus , 2pWrf n cleavage appeared to be prevented at concentrations of Ca which induced echinocytosis. In addition to proteolytic modifications, Ca2+ treatment also increased protein phosphorylation of at least 15 major cytoskeletal proteins, in a concentration-dependent manner. Similar changes were also observed when the erythrocytes were preincubated with ADR (10 PM) before calcium treatment. However, ADR appeared to lower the basal level of phosphorylation of cytoskeletal proteins in a concentration-dependent manner. The role of proteolysis and protein phosphorylatfon in the regulation of erythrocyte morphology will be discussed.
International
Reports,
Vol. 11, No. 3, March
The role proteins in the effect K.Y. Tang, Chemotherapy University,
of proteolysis and phosphorylation human erythrocytes discocyte-echinocyte of Rdriamycin. M.G. Thompson and J.A. Hickman, Pharmaceutical Sciences Group, Birmingham B4 7ET, U.K.
1987
of
249
cytoskeletal transition and
CRC Experimental Institute, Aston
The anthracycline antibiotic, Adrianlycin (ADR), is believed to exert its antitumour effect by intercalation of DNA. However, there is increasing evidence that ADR may exert its toxicity via In the present study, X interactions with the plasma membrane. human erythrocyte was used as a model to investigate the effect of Under various condiADR on membrane-cytoskeleton interactions. tions, human erythrocytes undergo.morphological transitions, which are believed to be regulated by the*+membrane and cytoskeleto in the presence of a Cahi llhen erythrocytes were treated with Ca they underwent concentration-dependent and ionophore (A231871, In the presence time-dependent discocyte-echinocyte transitions. of 1 mM calcium, the morphological transition to echinocytes could be observed as early as 30 seconds after the addition of 5 M SDS-PAGE analysis of the isolated membranes from Ca2+A23187. revealed the breakdown of two major treated erythrocytes cytoskeletal proteins: band 2.1 (ankyrin Mr 200,000) and band 4.1 (Mr 82,000). The breakdown of band 2.1 was indicated by the increase in band 2.3 (Mr 174,000) which is believed to be the proteolytic product of band 2.1. The increase in band 2.3 occurred in parallel with the morphological transition. The breakdown of band 4.1 was only observed after 30 mins treatment with 1 mM calcium. ADi)+was found capable of exerting a protective echinocytosis of human erythr effect agaiF?t the Ca -induced cytes at Ca concentrations of 0 to 150 ,M. Treatment with Ca8+ (150 pM) in the presence of the ionophore A23187 (5 ,JM), produced approximately 70-80% echinocytes after 5 mins. In contrast, there were only 15-20% echinocytes when erytp+ocytes were pre-incubated with ADR (10 MM) for 10 mins. At Ca concentrations of above 200 pM, pre-incubation with ADR had no significant effect on the formation of echinocytes. SDS-PAGE analysis indicated ADR had no significant effect on calcium-mediated cross-linking of cytoskeletal prote@s but inhibited the calcium-mediated breakdown of ankyrin at Ca concentrations of up to 400 u M. Thus , 2pWrf n cleavage appeared to be prevented at concentrations of Ca which induced echinocytosis. In addition to proteolytic modifications, Ca2+ treatment also increased protein phosphorylation of at least 15 major cytoskeletal proteins, in a concentration-dependent manner. Similar changes were also observed when the erythrocytes were preincubated with ADR (10 PM) before calcium treatment. However, ADR appeared to lower the basal level of phosphorylation of cytoskeletal proteins in a concentration-dependent manner. The role of proteolysis and protein phosphorylatfon in the regulation of erythrocyte morphology will be discussed.