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Clinical significance of serum calcineurin in acute leukemia
Clinica Chimica Acta 321 (2002) 17 – 21 www.elsevier.com/locate/clinchim
Clinical significance of serum calcineurin in acute leukemia S. Padma, C. Subramanyam* Department of Biochemistry, University College of Science, Osmania University, Hyderabad 500 007, India Received 11 December 2001; received in revised form 20 February 2002; accepted 28 February 2002
Abstract Background: Calcineurin is involved in T-lymphocyte activation as well as in the maturation of hematopoietic cells. Identification of this predominantly intracellular phosphatase and of calmodulin (CaM) in human sera warranted their assessment in different types of acute leukemias. Methods: Phosphatase activity of calcineurin (CaN) was assayed, involving the measurement of trifluoperazine-sensitive neutral phosphatase, in sera of leukemic patients before and after treatment. Calcineurin and calmodulin contents were also determined by ELISA employing monoclonal antibodies specific to the proteins. Results: The activity of calcineurin was decreased by 75% and 85% in sera of patients diagnosed either for acute lymphoid leukemia and acute myeloid leukemia, respectively, without apparent changes in calmodulin or calcineurin contents under both these conditions. In addition, the decreased calcineurin activity in acute myeloid leukemia was restored to levels comparable to non-leukemic individuals upon treatment. This was not observed in cases of acute lymphoid leukemia. Conclusions: These results suggest diagnostic utility in the measurement of serum calcineurin activity in acute leukemia. Restoration of normal calcineurin activity in patients undergoing treatment for acute myeloid leukemia may provide a means to monitor patient response to the prescribed therapeutic regimen. D 2002 Published by Elsevier Science B.V. Keywords: Acute lymphoid leukemia; Acute myeloid leukemia; Calcineurin; Calmodulin
1. Introduction Acute and chronic varieties of leukemia represent a group of malignant disorders that are characterized by infiltration of neoplastic blast cells of the hematopoietic system into the blood, bone marrow and other tissues. Conventional methods for distinguishing the myeloid variety of acute leukemia from the lymphoid variety involves evaluation of histopathological and immunological parameters related to (i) decreased red cell and platelet count (ii) structural and functional abnormal* Corresponding author. Tel.: +91-40-709-7044, +91-40-7091515; fax: +91-40-709-1515. E-mail address: [email protected] (C. Subramanyam).
ities of platelets and (iii) assaying the levels of serum immunoglobulins. However, the differential diagnosis between these two varieties of acute leukemia remains complex in view of the array of hematopoietic disorders involved in the etiology of acute leukemia. While the assay of enzymes such as terminal deoxynucleotidyl transferase (enhanced in acute lymphoid leukemia) [1] and lysozyme (enhanced in acute myeloid leukemia) [2] have been employed in this regard, there is a need to identify and develop simple, rapid diagnostic biochemical indicators of leukemic disease. Reports on the low expression of protein phosphatases, including protein phosphatase 1, 2A and 2B in leukemic blast cells (arrested at the stage of early pluripotent stem cells), and their modulation during the course of myelomonocytic commitment and maturation suggest
0009-8981/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 0 9 4 - 3
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S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
that assay of phosphatases may be of use in the diagnosis and prognosis of leukemia [3]. Earlier studies conducted in our laboratory demonstrated the presence of a protein that was immunologically akin to calcineurin (CaN; protein phosphatase 2 B; MW 80 kDa) in human sera [4]. CaN is the only neutral phosphatase regulated by ionic calcium and calmodulin (CaM) and is susceptible to inhibition by immunosuppressants such as cyclosporin and FK 506. While the role of CaN in regulating NFAT 1, involved in T-lymphocyte proliferation is well established [5], studies employing immunosuppressants demonstrated that increased expression of calcineurin may be involved in the inhibition of leukemic cell differentiation by 1, 25 dihydroxy vitamin D3 and all trans-retinoic acid [6,7]. In view of these reports, the activity as well as the content of CaN and CaM was assayed in sera of patients diagnosed with acute leukemia. The present study describes decreased activity of calcineurin, but not of its content, in leukemic cell lines as well as in sera of patients diagnosed for lymphoid or myeloid varieties of acute leukemia. The restoration of calcineurin activity in myeloid leukemia following treatment may also suggest clinical significance for calcineurin in the management of acute leukemia.
2. Materials Bovine serum albumin (BSA), morpholinoethane sulfonic acid (MES), calcineurin (bovine brain), calmodulin (bovine brain), monoclonal antibodies specific to calcineurin (a-subunit) or to bovine brain calmodulin, and trifluoperazine were from Sigma (St. Louis, MO). Goat anti-mouse IgG (conjugated to horseradish peroxidase) was from Bangalore Genei (Bangalore, India). Microtiter plates employed for ELISA (96 wells, flat bottom) were from Labsystems, France. All other chemicals were of analytical grade and obtained locally.
years) or acute myeloid leukemia (mean age 40 F 7 years) at local hospitals. Platelet count in such patients was < 100,000/mm3 and hemoglobin concentration ranged from 7 to 11 mg/dl. Serum samples were also collected from age-matched control individuals who were not on any medication and from leukemic patients undergoing chemotherapy either for acute lymphoid leukemia (treatment for 2 – 6 weeks with vincristine: 1.5 mg/m2/week, prednisone: 40 mg/m2/day and asparaginase: 10,000 U/m2/day) or for acute myeloid leukemia (treatment for 2– 6 weeks with cytosine arabinoside: 100 mg/m2/day and daunomycin: 10 mg/m2/ day). Response of leukemic patients to chemotherapy was ascertained by restoration of platelet count, decreased blast cell percentage to near normal values. 3.2. Assay of serum CaN activity After determining the protein contents by Bradford’s method [8], serum samples were assayed for calcineurin activity by determining the Ca2 + – CaMdependent, trifluoperazine-sensitive phosphatase activity according to the method described by Padma and Subramanyam [4]. Assays were conducted to measure p-nitrophenol released in the absence or in the presence of trifluoperazine (150 Amol/l) in a total volume of 100 Al containing 25 mmol/l Tris (pH 7.2), 25 mmol/l MES (pH 7.0), 2.4 Amol/l p-nitrophenyl phosphate, 1 mM MnCl2 and 30 Al of the sample ( c 30 Ag protein). Reactions were initiated by the addition of p-nitrophenyl phosphate, followed by incubation at 30 jC for 10 min. At the end of the incubation period, the reaction was terminated by the addition of 10 Al of 13% K2HPO4. Samples were then transferred to 96-well microtiter plates and absorbance measured at 405 nm in an ELISA reader (Spectra II, SLT Instruments, Kernenmunster, Austria). The difference between the amounts of p-nitrophenol released in the absence and in the presence of trifluoperazine indicated the activity of calcineurin. Units of enzyme activity are in terms of Amoles of p-nitrophenol formed per minute per deciliter.
3. Methods 3.1. Serum samples
3.3. Determination of calmodulin (CaM) and calcineurin (CaN) content by competitive ELISA
Samples of sera were collected from patients diagnosed with acute lymphoid leukemia (mean age 7 F 2
The CaM and CaN contents were determined in sera (diluted 1:5 with PBS) employing competitive
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
ELISA methods [4] in a total volume of 50 Al. The samples and the monoclonal antibodies (diluted 1 in 7500 for monoclonal anti CaN-a antibody and 1 in 1000 for monoclonal anti CaM in 0.01 mol/l PBS) were added onto 96-well, flat bottom polystyrene microtiter plates, which were previously coated with 0.5 ng/Al of CaN or 0.1 ng/Al of CaM, as the case may be, in carbonate – bicarbonate buffer (0.05 mol/l; pH 9.6). Nonspecific protein binding sites were blocked with 200 Al of 2% BSA prior to the addition of the samples and the monoclonal antibodies. After incubation (16 – 18 h at 4 jC for CaN and 2 h for CaM at 37 jC), the wells were washed with PBS containing 0.05% Tween-20 and incubated for a further period of 2 h at 37 jC with goat anti-mouse IgG labeled with horseradish peroxidase (1 in 2000 dilution). At the end of the incubation period, the wells were washed with PBS Tween 20 and incubated with 100 Al of substrate (4 mg of o-phenylene diamine mixed with 10 Al of H2O2 in 10 ml of citrate – phosphate buffer pH 5.0) for 1 h at 37 jC. The reaction was terminated with 8 mol/l H2SO4 and the color developed was read at 492 nm in an ELISA reader (Spectra II, SLT Instruments). 3.4. Statistics The results obtained were subjected to statistical analysis by Student’s t-test.
4. Results Results obtained are depicted in Table 1. The most significant observation of the study was the decreased activity of serum calcineurin in patients diagnosed with either acute lymphoid leukemia (75% decrease) or acute myeloid leukemia (85% decrease) in comparison to values obtained from controls. Even though such decreases were evident prior to the initiation of therapeutic treatment of the patients, the decreased activity of calcineurin was restored to normal values upon treatment of acute myeloid leukemia with cytosine arabinoside and daunomycin. Such an observation, however, could not be made in patients treated with vincristine, prednisone and asparaginase for acute lymphoid leukemia. On the contrary, quantitation of calcineurin content in sera of patients suffering
19
Table 1 Activity and content of calcineurin and calmodulin in leukemic sera Sample
CaN activitya CaN contentb CaM contentc [U/dl] [Ag/dl] [Ag/dl]
Acute lymphoid leukemia Control 16.8 F 6.9 (n = 10) 4.2 F 2.0 * (a) Before treatment (n = 12) (b) After 4.1 F 1.8 * treatment (n = 14)
209.5 F 33.4
48.8 F 14
152.2 F 27.9 * 64.8 F 10.8 * 156.6 F 31.7 * 67.6 F 11.6 *
Acute myeloid leukemia Control 11.8 F 1.81 214.0 F 84.5 25 F 5.4 (n = 10) (a) Before 1.5 F 0.47 * 175.5 F 46.2 * 26.8 F 9.01 treatment (n = 10) (b) After 12.1 F 3.7 ** 187 F 28.7 ** 41 F 15.1*** treatment (n = 11) a CaN activity was determined in the presence and absence of trifluoperazine employing p-nitrophenol as the substrate. One unit is defined as Amoles of p-nitrophenol formed/min/dl. b CaN contents were determined by the competitive ELISA protocol. c CaM contents were determined by the competitive ELISA protocol. * p > 0.001 is given in comparison to controls. ** p < 0.5 is given in comparison to controls. *** p < 0.2 is given in comparison to controls.
from either one of the acute leukemias did not demonstrate significant alterations in the content of calcineurin either before or during treatment. Simultaneous measurements of the contents of calmodulin—the activator of calcineurin—(to verify whether the observed decrease in serum calcineurin activity was due to limitation of calmodulin) did not reveal any significant changes in the calmodulin content. Interestingly, calcineurin activity was also decreased (77% decrease) in sera obtained from patients who were under treatment for chronic myeloid leukemia as compared to normal individuals (data not shown). As in case of acute lymphoid leukemia, such a decrease observed in chronic myeloid leukemia was unaccompanied either by significant decrease of calcineurin content in the sera or by restoration of calcineurin activity upon treatment. Thus, the restoration of serum calcineurin activity upon treatment of acute myeloid leukemia appears to be rather specific. Assaying the
20
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
calcineurin content and activity in leukemia cell lines of lymphoid (Molt-4) and myeloid origin (HL-60) also substantiated the above results. While no change could be observed in the content of calcineurin, its activity was decreased in both these cell lines without any apparent decrease in the calmodulin content (Data not shown).
5. Discussion Calmodulin-mediated second messenger functions of Ca2 + are well recognized to play an important role in cell growth through specific kinases and phosphatases that target intracellular proteins [9– 11]. In recent years, significant evidences are becoming available to indicate that the activity of Ca2 + -calmodulin-mediated protein phosphorylation and dephosphorylation are subject to regulation by intracellular redox state as well as by oxidants and antioxidants [12,13]. Among the serine/threonine phosphatases, it has been reported that the activity of calcineurin was affected by oxidants such as H2O2[14]. Upon binding with Ca2 + and calmodulin, the Fe3 + – Zn2 + catalytic center of calcineurin becomes susceptible to oxidative attack by superoxide anion. Protection of this bimetallic center of calcineurin, from oxidation, by superoxide dismutase is an important aspect of Ca2 + -dependent regulation of cellular processes to be modulated by the redox potential [15]. In view of such reports, it was of interest to assay the calcineurin activity in acute leukemias. A salient observation made during the study relates to the decreased activity of calcineurin (75 – 85% decrease), unaccompanied with decrease in its content or in the content of calmodulin, in the sera of patients diagnosed for acute leukemia. The restoration of normal calcineurin activity in sera of patients under treatment for acute myeloid leukemia also suggests a putative prognostic role for calcineurin in monitoring the patient responsiveness to the recommended chemotherapy with cytosine arabinoside and daunomycin under such conditions. The present results assume significance in view of earlier reports on (i) inhibition of the phosphatase activity of calcineurin by oxidants such as H2O2, superoxide and glutathione disulfide in a dose-dependent manner [16], and (ii) oxidative stressinduced apoptosis in the leukemia cell line, HL-60 [13,17]. Further, decrease in the phosphatase activity
of calcineurin in two T-cell leukemic cell lines (Warzburg and Jurkat cell lines) was associated with an increase in hydrogen peroxide production [18]. Recent studies from our laboratory further demonstrated a putative role for calcineurin in lymphopenia associated with chronic renal failure [19]. From the results presented above and the increasing evidence for the susceptibility of calcineurin activity to oxidative damage, it may be concluded that alterations observed in serum calcineurin activity in patients diagnosed for different acute leukemias may provide a probable diagnostic biochemical marker in the differential diagnosis of acute leukemia. Acknowledgements This work was supported by the Lady Tata Memorial Trust through a postdoctoral fellowship awarded to Dr. S. Padma. Thanks are also due to DST for the award of research grant no. SP/SO/A69/97 to CS. References [1] Sujimoto M, Bollom FJ. Terminal deoxy nucleotidyl transferase (Tdt) in chick embryo lymphoid tissues. J Immunol 1979; 122:392 – 7. [2] Weinstein HJ. Acute leukemias. In: Wyngaarden JB, Smith LH, editors. Cecil textbook of medicine. Philadelphia, PA: Saunders, 1982. pp. 920 – 6. [3] Yamamoto M, Suzuki Y, Kihira H, et al. Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells. Leukemia 1999;13:595 – 600. [4] Padma S, Subramanyam C. Extracellular calcineurin: identification and quantitation in serum and amniotic fluid. Clin Biochem 1999;32:491 – 4. [5] Luo C, Burgeon E, Carew JA, et al. Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. Mol Cell Biol 1996; 16:3955 – 66. [6] Omay SB, Nakai K, Kuno T, Shiku H, Nishikawa M. 1a25, Dihydroxy D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation. Blood 1996;87:2947 – 55. [7] Kihira T, Hiasa A, Yamamoto M, Katayama N, et al. Possible involvement of calcineurin in retinoic acid induce inhibition of leukemic HL-60 cell proliferation. Int J Oncol 1998;12:629 – 34. [8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248 – 54. [9] Ingerbritsen TS, Stewart AA, Cohen P. The protein phosphatases involved in cellular regulation. Measurement of type I and type 2 protein phosphatases in extracts of mammalian
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
[10]
[11]
[12] [13]
[14]
tissue; An assessment of their physiological roles. Eur J Biol 1983;89:379. Subramanyam C, Honn SC, Reed WC, Reddy GPV. Nuclear localization of 68 kDa calmodulin binding protein is associated with the onset of DNA replication. J Cell Physiol 1990; 144:423 – 8. Reddy G, Reed WC, Sheehan E, Sacks D. Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells. Biochemistry 1992;31:10426 – 30. Suzuki JY, Forman JH, Sevanian A. Oxidants as stimulators of signal transduction. Free Radical Biol Med 1997;22:269 – 85. Sommer D, Fakata KL, Swanson SA, Stemmer PM. Modulation of the phosphatase activity of calcineurin by oxidants and antioxidants in vitro. Eur J Biochem 2000;267:2312 – 22. Reiter TA, Abraham RT, Choi M, Rusnak F. Redox regulation of calcineurin in T-lymphocytes. J Biol Inorg Chem 1999; 4:632 – 44.
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[15] Wang X, Culotta VC, Klee CB. Superoxide dismutase protects calcineurin from inactivation. Nature 1996;383:434 – 7. [16] Lee JE, Sohn J, Lee KC, Son CS, Tockgo YC. Regulation of bcl-2 family in hydrogen peroxide-induced apoptosis in human leukemia HL-60 cells. Exp Mol Med 2000;32:42 – 6. [17] Jing Y, Dai J, Chalmers-Redman RM, Tatton WG, Waxman S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide dependent pathway. Blood 1999;94:2101 – 11. [18] Schoene NW, Kamara KS. Population doubling time, phosphatase activity and hydrogen peroxide generation in Jurkat cells. Free Radical Biol Med 1999;27:364 – 9. [19] Sasikala M, Sadasivudu B, Subramanyam C. A putative role for calcineurin in lymphopenia associated with chronic renal failure. Clin Biochem 2000;33:691 – 4.
Clinical significance of serum calcineurin in acute leukemia S. Padma, C. Subramanyam* Department of Biochemistry, University College of Science, Osmania University, Hyderabad 500 007, India Received 11 December 2001; received in revised form 20 February 2002; accepted 28 February 2002
Abstract Background: Calcineurin is involved in T-lymphocyte activation as well as in the maturation of hematopoietic cells. Identification of this predominantly intracellular phosphatase and of calmodulin (CaM) in human sera warranted their assessment in different types of acute leukemias. Methods: Phosphatase activity of calcineurin (CaN) was assayed, involving the measurement of trifluoperazine-sensitive neutral phosphatase, in sera of leukemic patients before and after treatment. Calcineurin and calmodulin contents were also determined by ELISA employing monoclonal antibodies specific to the proteins. Results: The activity of calcineurin was decreased by 75% and 85% in sera of patients diagnosed either for acute lymphoid leukemia and acute myeloid leukemia, respectively, without apparent changes in calmodulin or calcineurin contents under both these conditions. In addition, the decreased calcineurin activity in acute myeloid leukemia was restored to levels comparable to non-leukemic individuals upon treatment. This was not observed in cases of acute lymphoid leukemia. Conclusions: These results suggest diagnostic utility in the measurement of serum calcineurin activity in acute leukemia. Restoration of normal calcineurin activity in patients undergoing treatment for acute myeloid leukemia may provide a means to monitor patient response to the prescribed therapeutic regimen. D 2002 Published by Elsevier Science B.V. Keywords: Acute lymphoid leukemia; Acute myeloid leukemia; Calcineurin; Calmodulin
1. Introduction Acute and chronic varieties of leukemia represent a group of malignant disorders that are characterized by infiltration of neoplastic blast cells of the hematopoietic system into the blood, bone marrow and other tissues. Conventional methods for distinguishing the myeloid variety of acute leukemia from the lymphoid variety involves evaluation of histopathological and immunological parameters related to (i) decreased red cell and platelet count (ii) structural and functional abnormal* Corresponding author. Tel.: +91-40-709-7044, +91-40-7091515; fax: +91-40-709-1515. E-mail address: [email protected] (C. Subramanyam).
ities of platelets and (iii) assaying the levels of serum immunoglobulins. However, the differential diagnosis between these two varieties of acute leukemia remains complex in view of the array of hematopoietic disorders involved in the etiology of acute leukemia. While the assay of enzymes such as terminal deoxynucleotidyl transferase (enhanced in acute lymphoid leukemia) [1] and lysozyme (enhanced in acute myeloid leukemia) [2] have been employed in this regard, there is a need to identify and develop simple, rapid diagnostic biochemical indicators of leukemic disease. Reports on the low expression of protein phosphatases, including protein phosphatase 1, 2A and 2B in leukemic blast cells (arrested at the stage of early pluripotent stem cells), and their modulation during the course of myelomonocytic commitment and maturation suggest
0009-8981/02/$ - see front matter D 2002 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 8 9 8 1 ( 0 2 ) 0 0 0 9 4 - 3
18
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
that assay of phosphatases may be of use in the diagnosis and prognosis of leukemia [3]. Earlier studies conducted in our laboratory demonstrated the presence of a protein that was immunologically akin to calcineurin (CaN; protein phosphatase 2 B; MW 80 kDa) in human sera [4]. CaN is the only neutral phosphatase regulated by ionic calcium and calmodulin (CaM) and is susceptible to inhibition by immunosuppressants such as cyclosporin and FK 506. While the role of CaN in regulating NFAT 1, involved in T-lymphocyte proliferation is well established [5], studies employing immunosuppressants demonstrated that increased expression of calcineurin may be involved in the inhibition of leukemic cell differentiation by 1, 25 dihydroxy vitamin D3 and all trans-retinoic acid [6,7]. In view of these reports, the activity as well as the content of CaN and CaM was assayed in sera of patients diagnosed with acute leukemia. The present study describes decreased activity of calcineurin, but not of its content, in leukemic cell lines as well as in sera of patients diagnosed for lymphoid or myeloid varieties of acute leukemia. The restoration of calcineurin activity in myeloid leukemia following treatment may also suggest clinical significance for calcineurin in the management of acute leukemia.
2. Materials Bovine serum albumin (BSA), morpholinoethane sulfonic acid (MES), calcineurin (bovine brain), calmodulin (bovine brain), monoclonal antibodies specific to calcineurin (a-subunit) or to bovine brain calmodulin, and trifluoperazine were from Sigma (St. Louis, MO). Goat anti-mouse IgG (conjugated to horseradish peroxidase) was from Bangalore Genei (Bangalore, India). Microtiter plates employed for ELISA (96 wells, flat bottom) were from Labsystems, France. All other chemicals were of analytical grade and obtained locally.
years) or acute myeloid leukemia (mean age 40 F 7 years) at local hospitals. Platelet count in such patients was < 100,000/mm3 and hemoglobin concentration ranged from 7 to 11 mg/dl. Serum samples were also collected from age-matched control individuals who were not on any medication and from leukemic patients undergoing chemotherapy either for acute lymphoid leukemia (treatment for 2 – 6 weeks with vincristine: 1.5 mg/m2/week, prednisone: 40 mg/m2/day and asparaginase: 10,000 U/m2/day) or for acute myeloid leukemia (treatment for 2– 6 weeks with cytosine arabinoside: 100 mg/m2/day and daunomycin: 10 mg/m2/ day). Response of leukemic patients to chemotherapy was ascertained by restoration of platelet count, decreased blast cell percentage to near normal values. 3.2. Assay of serum CaN activity After determining the protein contents by Bradford’s method [8], serum samples were assayed for calcineurin activity by determining the Ca2 + – CaMdependent, trifluoperazine-sensitive phosphatase activity according to the method described by Padma and Subramanyam [4]. Assays were conducted to measure p-nitrophenol released in the absence or in the presence of trifluoperazine (150 Amol/l) in a total volume of 100 Al containing 25 mmol/l Tris (pH 7.2), 25 mmol/l MES (pH 7.0), 2.4 Amol/l p-nitrophenyl phosphate, 1 mM MnCl2 and 30 Al of the sample ( c 30 Ag protein). Reactions were initiated by the addition of p-nitrophenyl phosphate, followed by incubation at 30 jC for 10 min. At the end of the incubation period, the reaction was terminated by the addition of 10 Al of 13% K2HPO4. Samples were then transferred to 96-well microtiter plates and absorbance measured at 405 nm in an ELISA reader (Spectra II, SLT Instruments, Kernenmunster, Austria). The difference between the amounts of p-nitrophenol released in the absence and in the presence of trifluoperazine indicated the activity of calcineurin. Units of enzyme activity are in terms of Amoles of p-nitrophenol formed per minute per deciliter.
3. Methods 3.1. Serum samples
3.3. Determination of calmodulin (CaM) and calcineurin (CaN) content by competitive ELISA
Samples of sera were collected from patients diagnosed with acute lymphoid leukemia (mean age 7 F 2
The CaM and CaN contents were determined in sera (diluted 1:5 with PBS) employing competitive
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
ELISA methods [4] in a total volume of 50 Al. The samples and the monoclonal antibodies (diluted 1 in 7500 for monoclonal anti CaN-a antibody and 1 in 1000 for monoclonal anti CaM in 0.01 mol/l PBS) were added onto 96-well, flat bottom polystyrene microtiter plates, which were previously coated with 0.5 ng/Al of CaN or 0.1 ng/Al of CaM, as the case may be, in carbonate – bicarbonate buffer (0.05 mol/l; pH 9.6). Nonspecific protein binding sites were blocked with 200 Al of 2% BSA prior to the addition of the samples and the monoclonal antibodies. After incubation (16 – 18 h at 4 jC for CaN and 2 h for CaM at 37 jC), the wells were washed with PBS containing 0.05% Tween-20 and incubated for a further period of 2 h at 37 jC with goat anti-mouse IgG labeled with horseradish peroxidase (1 in 2000 dilution). At the end of the incubation period, the wells were washed with PBS Tween 20 and incubated with 100 Al of substrate (4 mg of o-phenylene diamine mixed with 10 Al of H2O2 in 10 ml of citrate – phosphate buffer pH 5.0) for 1 h at 37 jC. The reaction was terminated with 8 mol/l H2SO4 and the color developed was read at 492 nm in an ELISA reader (Spectra II, SLT Instruments). 3.4. Statistics The results obtained were subjected to statistical analysis by Student’s t-test.
4. Results Results obtained are depicted in Table 1. The most significant observation of the study was the decreased activity of serum calcineurin in patients diagnosed with either acute lymphoid leukemia (75% decrease) or acute myeloid leukemia (85% decrease) in comparison to values obtained from controls. Even though such decreases were evident prior to the initiation of therapeutic treatment of the patients, the decreased activity of calcineurin was restored to normal values upon treatment of acute myeloid leukemia with cytosine arabinoside and daunomycin. Such an observation, however, could not be made in patients treated with vincristine, prednisone and asparaginase for acute lymphoid leukemia. On the contrary, quantitation of calcineurin content in sera of patients suffering
19
Table 1 Activity and content of calcineurin and calmodulin in leukemic sera Sample
CaN activitya CaN contentb CaM contentc [U/dl] [Ag/dl] [Ag/dl]
Acute lymphoid leukemia Control 16.8 F 6.9 (n = 10) 4.2 F 2.0 * (a) Before treatment (n = 12) (b) After 4.1 F 1.8 * treatment (n = 14)
209.5 F 33.4
48.8 F 14
152.2 F 27.9 * 64.8 F 10.8 * 156.6 F 31.7 * 67.6 F 11.6 *
Acute myeloid leukemia Control 11.8 F 1.81 214.0 F 84.5 25 F 5.4 (n = 10) (a) Before 1.5 F 0.47 * 175.5 F 46.2 * 26.8 F 9.01 treatment (n = 10) (b) After 12.1 F 3.7 ** 187 F 28.7 ** 41 F 15.1*** treatment (n = 11) a CaN activity was determined in the presence and absence of trifluoperazine employing p-nitrophenol as the substrate. One unit is defined as Amoles of p-nitrophenol formed/min/dl. b CaN contents were determined by the competitive ELISA protocol. c CaM contents were determined by the competitive ELISA protocol. * p > 0.001 is given in comparison to controls. ** p < 0.5 is given in comparison to controls. *** p < 0.2 is given in comparison to controls.
from either one of the acute leukemias did not demonstrate significant alterations in the content of calcineurin either before or during treatment. Simultaneous measurements of the contents of calmodulin—the activator of calcineurin—(to verify whether the observed decrease in serum calcineurin activity was due to limitation of calmodulin) did not reveal any significant changes in the calmodulin content. Interestingly, calcineurin activity was also decreased (77% decrease) in sera obtained from patients who were under treatment for chronic myeloid leukemia as compared to normal individuals (data not shown). As in case of acute lymphoid leukemia, such a decrease observed in chronic myeloid leukemia was unaccompanied either by significant decrease of calcineurin content in the sera or by restoration of calcineurin activity upon treatment. Thus, the restoration of serum calcineurin activity upon treatment of acute myeloid leukemia appears to be rather specific. Assaying the
20
S. Padma, C. Subramanyam / Clinica Chimica Acta 321 (2002) 17–21
calcineurin content and activity in leukemia cell lines of lymphoid (Molt-4) and myeloid origin (HL-60) also substantiated the above results. While no change could be observed in the content of calcineurin, its activity was decreased in both these cell lines without any apparent decrease in the calmodulin content (Data not shown).
5. Discussion Calmodulin-mediated second messenger functions of Ca2 + are well recognized to play an important role in cell growth through specific kinases and phosphatases that target intracellular proteins [9– 11]. In recent years, significant evidences are becoming available to indicate that the activity of Ca2 + -calmodulin-mediated protein phosphorylation and dephosphorylation are subject to regulation by intracellular redox state as well as by oxidants and antioxidants [12,13]. Among the serine/threonine phosphatases, it has been reported that the activity of calcineurin was affected by oxidants such as H2O2[14]. Upon binding with Ca2 + and calmodulin, the Fe3 + – Zn2 + catalytic center of calcineurin becomes susceptible to oxidative attack by superoxide anion. Protection of this bimetallic center of calcineurin, from oxidation, by superoxide dismutase is an important aspect of Ca2 + -dependent regulation of cellular processes to be modulated by the redox potential [15]. In view of such reports, it was of interest to assay the calcineurin activity in acute leukemias. A salient observation made during the study relates to the decreased activity of calcineurin (75 – 85% decrease), unaccompanied with decrease in its content or in the content of calmodulin, in the sera of patients diagnosed for acute leukemia. The restoration of normal calcineurin activity in sera of patients under treatment for acute myeloid leukemia also suggests a putative prognostic role for calcineurin in monitoring the patient responsiveness to the recommended chemotherapy with cytosine arabinoside and daunomycin under such conditions. The present results assume significance in view of earlier reports on (i) inhibition of the phosphatase activity of calcineurin by oxidants such as H2O2, superoxide and glutathione disulfide in a dose-dependent manner [16], and (ii) oxidative stressinduced apoptosis in the leukemia cell line, HL-60 [13,17]. Further, decrease in the phosphatase activity
of calcineurin in two T-cell leukemic cell lines (Warzburg and Jurkat cell lines) was associated with an increase in hydrogen peroxide production [18]. Recent studies from our laboratory further demonstrated a putative role for calcineurin in lymphopenia associated with chronic renal failure [19]. From the results presented above and the increasing evidence for the susceptibility of calcineurin activity to oxidative damage, it may be concluded that alterations observed in serum calcineurin activity in patients diagnosed for different acute leukemias may provide a probable diagnostic biochemical marker in the differential diagnosis of acute leukemia. Acknowledgements This work was supported by the Lady Tata Memorial Trust through a postdoctoral fellowship awarded to Dr. S. Padma. Thanks are also due to DST for the award of research grant no. SP/SO/A69/97 to CS. References [1] Sujimoto M, Bollom FJ. Terminal deoxy nucleotidyl transferase (Tdt) in chick embryo lymphoid tissues. J Immunol 1979; 122:392 – 7. [2] Weinstein HJ. Acute leukemias. In: Wyngaarden JB, Smith LH, editors. Cecil textbook of medicine. Philadelphia, PA: Saunders, 1982. pp. 920 – 6. [3] Yamamoto M, Suzuki Y, Kihira H, et al. Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells. Leukemia 1999;13:595 – 600. [4] Padma S, Subramanyam C. Extracellular calcineurin: identification and quantitation in serum and amniotic fluid. Clin Biochem 1999;32:491 – 4. [5] Luo C, Burgeon E, Carew JA, et al. Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. Mol Cell Biol 1996; 16:3955 – 66. [6] Omay SB, Nakai K, Kuno T, Shiku H, Nishikawa M. 1a25, Dihydroxy D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation. Blood 1996;87:2947 – 55. [7] Kihira T, Hiasa A, Yamamoto M, Katayama N, et al. Possible involvement of calcineurin in retinoic acid induce inhibition of leukemic HL-60 cell proliferation. Int J Oncol 1998;12:629 – 34. [8] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248 – 54. [9] Ingerbritsen TS, Stewart AA, Cohen P. The protein phosphatases involved in cellular regulation. Measurement of type I and type 2 protein phosphatases in extracts of mammalian
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