When cells are exposed to a variety of stimuli, there is increased expression of stress or heat shock proteins, a major representative of which is hsp70. The objective of the present work was to investigate the endogenous expression of hsp70 in peripheral blood mononuclear cells and erythrocytes of patients suffering from b-thalassaemia and to correlate hsp70 levels with patients' antioxidant status. Blood samples were obtained from thalassaemia major patients aged 16-24 years. Hsp70 was identified with a mouse monoclonal anti-human hsp70 antibodies using the Western blot procedure. The total antioxidant status was determined by means of a commercial kit of RANDOX. It was established that: i) Hsp70 levels were low in mononuclear cells, ii) Hsp70 was not appreciably induced by incubation at 43°C, Hi) in erythrocytes, however, there was a marked endogenous expression ofhsp70 - thalassaemics express more hsp70 than control subjects, iv) the antioxidant status of thalassaemics was by about 20 % less than the control one. The increased endogenous hsp70 in thalassaemic erythrocytes was consistent with the hypothesis that the elevated levels of denaturated globins induced the expression of stress proteins during erythopoiesis. We are currently investigating whether there is a correlation between the severity of the clinical symptoms and hsp70 levels

Bonanou-Tzedaki, S.

Kolettas, E.

Kovachev, D.

English

Varna Medical University Press: Journals

Scripta Scientifica Medica, vol. 30 (1998), pp. 83-87 Copyright © Medical University, Varna STUDY OF HEAT SHOCK PROTEINS IN THALASSAEMIC PATIENTS D. Kovachev, E . Kolettas*, S. Bonanou-Tzedaki* department of Biology, Medical University of Varna, Varna, Bulgaria and ^Laboratory of Biochemistry, School of Medicine, University of Thessaly, Larissa, Greece When cells are exposed to a variety of stimuli, there is increased expression of stress or heat shock proteins, a major representative of which is hsp70. The objective of the present work was to investigate the endogenous expression of hsp70 in peripheral blood mono­nuclear cells and erythrocytes of patients suffering from b-thalassaemia and to correlate hsp70 levels with patients' antioxidant status. Blood samples were obtained from thalassaemia major patients aged 16-24 years. Hsp70 was identified with a mouse mono­clonal anti-human hsp70 antibodies using the Western blot procedure. The total antioxi­dant status was determined by means of a commercial kit of RANDOX. It was established that: i) Hsp70 levels were low in mononuclear cells, ii) Hsp70 was not appreciably induced by incubation at 43°C, Hi) in erythrocytes, however, there was a marked endogenous ex­pression ofhsp70 - thalassaemics express more hsp70 than control subjects, iv) the antioxi­dant status of thalassaemics was by about 20 % less than the control one. The increased endogenous hsp70 in thalassaemic erythrocytes was consistent with the hypothesis that the elevated levels of denaturated globins induced the expression of stress proteins during erythopoiesis. We are currently investigating whether there is a correlation between the severity of the clinical symptoms and hsp70 levels. blood cell transfusions combat the anaemia but result in accumulation of tissue iron which if untreated is fatal in the second decade of life. The develop­ment of iron chelation therapy has led to dramatic improvements in survival and quality of life. Iron overload is, however, still a major concern. Under physiological conditions ferric ions are bound to proteins (transferrin and fer­ritin) and are not available to catalyse the conversion of molecular oxygen to Key-words: Beta-thalassaemia, heat shock proteins, hsp70, antioxidant status, hae­moglobin, erythrocytes Homozygous b-thalassaemia is haracterized by absent or reduced pro­duction of haemoglobin b-chains. The haemoglobin а-chains which remain in excess aggregate in insoluble homotetr-amers which precipitate intracellular^ leading to premature red cell destruc­tion (haemolytic anaemia). Regular red Address for correspondence: D. Kovachev, Dept. of Biology, Medical University, 55 Marin Drinov St, BG-9002 Varna, BULGARIA E-mail: biologie@asclep.muvar.acad.bg D. Kovachev, Е. Kolettas, S. Bonanou-Tzedaki reactive oxygen species ( R O S ) , e. g., hydroxyl ions, superoxide and hydro­gen peroxide. Under conditions of iron overload such as thalassaemia, how­ever, there is an increase of low-molecu­lar weight iron w h i c h promotes peroxidative damage and cell injury (4,5) and causes a decreased total anti­oxidant status of the patient (10). When cells are exposed to a variety of stimuli (physiopathologic conditions and environmental assaults, including oxidative stress), there is an increased expression of certain classes of proteins, the so-called stress or heat shock proteins, a major representative of which is hsp70, which protect cellu­lar proteins from denaturation (8). The human hsp70 family includes 11 genes expressed either const i tu t ively (hsp70c), or after induction by various agents (hsp70i). Although the expres­sion of hsp70 has been studied in vari­ous haemopoietic cells, there is no in­formation about its expression in the white and red blood cells of patients suf­fering from iron overload. We reasoned that the increased iron levels found in b-thalassaemia would result in in­creased R O S formation and expression of hsp70. This stress response might confer considerable protection to red blood cells because it could decrease the effective concentration of free hae­moglobin а-chains through association with hsp70, and to white blood cells which may become more able to with­stand the damaging effects of R O S . The objective of the present study was to investigate the endogenous expression of hsp70 in peripheral blood mononuclear cells and erythrocytes of patients suffering from b-thalassaemia and to correlate hsp70 levels with pa­tients' antioxidant status. MATERIAL AND METHODS Blood samples were obtained from patients aged 16-24 years and suf­fering from thalassaemia major who were on Desferrioxamine treatment just before blood transfusion. Samples from age-matched blood donors were used as normal controls. Peripheral blood mononuclear cells and erythrocytes were obtained by density centrifugation of whole blood-EDTA samples through F i c o l l . The cel ls were extensively washed with H A N K S and then resus-pended in R P M I 1640 containing 10 % fetal calf serum, 1 % glutamine, and penicillin-streptomycin. The cells were preincubated at 37°C for 2 hours, heat-shocked at 43°C for 2 hours, and al­lowed to recover at 37°C for 1 hour. Af­ter pelleting and washing, they were ly-sed in triple detergent buffer, boiled in x2 Laemmli sample buffer (6) and the proteins were separated by S D S - P A G E and transferred to nitrocellulose filters. Hsp70 was identified by probing with a mouse monoclonal anti-human hsp70 antibody (Sigma H5147) which detects both constitutive and inducible forms. Colour was developed after the addi-84 Study of heat shock proteins . n of a reporter anti-mouse IgG-HPR antibody using a horseradish peroxidase chromogen solution. Human hsp70 (Sigma) was used as positive control. Patients' total antioxidant status was de­termined in serum samples by follow­ing the absorption of the radical cation A B T S (10) using a kit by R A N D O X . I ther clinical chemical analyses were performed at the Larissa Hospital D i ­agnostic Laboratories, Larissa. ESULTS AND DISCUSSION Fig. 1 shows the expression of hsp70 in the white and red blood cells of eight thalassaemic and six control subjects. They are representative of the Western blots obtained with several dif-ferent samples. В 1 2 3 4 5 6 7 8 9 10 11 12 W h i t e ce l l s R e d cel ls Fig. 1. Expression ofhsp70 in white and red blood cells of normal and thalassaemic subjects We find out that in peripheral blood mononuclear cells: i ) endogenous hsp70 levels, i i ) hsp70 is not apprecia­bly induced by incubation at 43°C, i i i ) thalassaemic cells express even less hsp70 than control ones when equal amounts of proteins are loaded, iv ) R N A extracted from mononuclear cells of normal and thalassaemic subjects also give a very faint signal for hsp70m R N A on Northern blots (results not shown). In erythrocytes, however, there is a marked endogenous expression of hsp70. Thalassaemics express more hsp70 than control subjects. The total antioxidant status of the serum of thalassaemic subjects is by approximately 20 % less than that of normal subjects (Table 1). Table 1 Total antioxidant status of control and thalassaemic subjects (Each value represents the mean ±SD) Subjects m M / L Control (n=8) 1,49 ±0,17 Thalassaemic (n=8) 1,21 ±0,16 (p < 0,1 by Student's Mest). The mean values of selected haematologic indices for the thalassaemic and control individuals are the following (Table 2): Table 2 Haematologic parameters of control and thalassaemic subjects Parameters T С g % haemoglobin 10,2 14,3 g/mliron 260,0 110,0 ng/ml ferritin 1,890 82 85 D. Kovachev, Е. Kolettas, S. Bonanou-Tzedaki In haemopoietic cells the ex­pression of hsp70 has been studied in chicken, rabbit, murine and human cells and in various leukaemic cell lines (2,3). It has been found that monocytes and granulocytes, but not lymphocytes, constitutively express hsp70 (2). Act i ­vation of either lymphocytes or mono­cytes by cytokines or exposure to oxi­dative stress leads to induction or in­creased expression of hsp70 (9). Its ex­pression is induced during erythroid cell differentiation (1) and increases further by heat shock. Our results with the mono­nuclear cells (which consist of approxi­mately 85 % lymphocytes and 15 % monocytes) agree with the findings in the literature available about the low levels of endogenous hsp70 expression. The fact that we could not find any in­crease of hsp70 levels in the thalassaemic white cells was unex­pected, given increased patients' iron and ferritin levels and the decreased serum antioxidant capacity. It suggests that the presumed oxidative stress either does not affect mononuclear cells, or is compensated by other mecha­nisms. The increased endogenous hsp70 in thalassaemic erythrocytess, however, is consistent with the hypothesis that the elevated levels of denaturated globins induce the expression of stress proteins during erythropoiesis. Finally, this 20 % decrease of the antioxidant status of the thalassaemic patients is comparable to the 14 % one reported in another study (7). We are currently investigating whether there is a correlation between the severity of the clinical symptoms and hsp70 levels. Acknowledgements: We thank the University of Thessaly Research Committee for financial assistance and Drs Halkias and Vagenas for providing the blood samples. REFERENCES 1. B e i - P a r a s k e v o p o u l o u , T . A . , S . A . B o n a n o u - T z e d a k i , H . V. A r n s t e i n . Biochem. Soc. Trans., 14, 1986, 973-977.-2. C I e r g e t , M . , B . S . P o l l a . Proc. Natl. Acad. Sci. USA, 87, 1990, 1081-1090.-3. F i n c a t o , G . , et a l . Blood, 11, 1991,579-588.- 4. H a l l i w e l l , В . , J . M . C . G u t t e r i d g e . Meth. Enzymol., 186, 1990,1-8.-5.Hershko, C , G . G r a h a m , G . W. B a t e s , E . A . R a c h m i l e w i t z . Brit. J. Haematol, 40, 1978, 255-261.- 6. L a e m m l i , U . K . Nature, 111, 1990, 680-6 7 4 . - 7 . L i v r e a , M . A . , et a l . Blood, 88,1996, 3608-3614.- 8. M o r i m o t o , R . , A . T i s s i e r e s , C . G e o r g o p o u l o s , Eds. TheBiology of Heat Shock Proteins, New York, Cold Spring Harbor Laboratory, 1990 . -9 .0 'Fa r r e l l , F . J . , В . M . H a n n i g a n , Y . A . B a r n e t t . Biochem. Soc. Trans., 24, 1996, 718-722.- 10. R i c e - E v a n s , C . N . J . M i l l e r . Meth. Enzymol., 234, 1994, 279-290. 86 Study of heat shock proteins 87 И з с л е д в а н е на т о п л и н н о - ш о к о в и п р о т е и н и п р и б о л н и с т а л а с е м и я Д. Ковачев, Е. Колетас*, С. Бонану-Цедаки* Катедра по биология, Медицински Университет - Варна, България и * Катедра по биохимия, Медицински факултет, Тесалийски Университет - Лариса, Гърция Резюме: Когато клетката се подложи на редица външни и вътрешни въздействия рязко се увеличава експресията на т.н. стресови или топлинно-шокови протеини (ТШП). Счита се, че главна роля тук играят ТШП-70. Цел на настоящото изследване бе да се докаже ендогенната поява на ТШП-70 в еритроцити и мононуклеарни клетки на болни с b-таласемия. Проби от кръвта на болни на възраст от 16 до 24 г. бяха получени от градската болница в Лариса, Гърция. ТШП-70 бяха идентифицирани чрез моноклонални антитела. Антиоксидантният статус на болните бе определен с помощта на кит от фирмата RANDOX. Получените резултати показват, че: 1) нивото на ТШП-70 в мононуклеарните клетки е много ниско; 2) допълнителният топлинен шок на клетките при 43°С не повишава значимо изходното ниво; 3) при еритроцитите се наблюдава значителна експресия на ТШП-70 в сравнение с контролата, 4) антиоксидантният статус на болните е с около 20 % по-нисък в сравнение с контролата. Повишеното ниво на ТШП-70 в таласемични червени кръвни клетки е в съзвучие с хипотезата, че големите количества денатуриран глобин може да индуцира експресията на стресови протеини по време на еритропоезата. В ход са сравнителни изследвания между тежестта на клиничната картина и нивата на ТШП-70 при болни с таласемия. 

STUDY OF HEAT SHOCK PROTEINS IN THALASSAEMIC PATIENTS

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STUDY OF HEAT SHOCK PROTEINS IN THALASSAEMIC PATIENTS

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