Scand J Haematol (1977) 19, 207-210
Heterogeneity of Acid Phosphatase,
p - Glucuronidase, and a -Naphthyl-Acetate Esterase in Normal and Leukaemic Lymphocytes Aleksander Celidski #&Wladyslaw Pajdak Department of Clinical Chemistry, Institute of Internal Medicine, Medical Academy Cracow, Poland
Comparative study of isocnzymic patterns of acid phosphatase, P-glucuronidase and a-naphthyl acetate esterase of normal and chronic lymphocytic leukaemia (CLL) lymphocytes was carried out by means of polyacrylamide gel electrophoresis. The isoenzymograms of acid phosphatase and P-glucuronidase of leukaemic cells were similar to that of normal lymphocytes. However, different patterns were found in the case of a-naphthyl acetate esterase. Two prominent anodal bands were present in CLL lymphocytes; these bands were either absent or very weak in normal cells. Key words: chr. lymphocytic leukaemia - isoenzymes - lysosyme hydrolase polyacrylamide gel electrophoresis
-
Accepted for publication April 18, 1977 Correspondence to: Dr. Aleksander Celihski, Department o'f Clinical Chemistry, Institute of Internal Medicine, Kopernika 17, 31-501 Krakow, Poland
Despite many comparative cytochemical studies on normal and leukaemic lymphocytes, and extensive studies on blastic transformation in culture of both normal and CLL cells, information concerning the isoenzymic pattems of lymphocytic enzymes is scanty. It is not excluded that CLL may be caused by disturbances of lysosomal enzymes (Allison 1969) on the other hand, the disease may cause changes in activities of these enzymes (Douglas et al 1973). Hence, information on the shifts in isoenzymograms seems to be of importance for better understanding of the leukaemic process.
The purpose of current repo'rt is to compare the isoenzymic patterns of 3 enzymes of nolrmal and CLL lymphocytes. MATERIALS AND METHODS Normal and CLL lymphocytes were isolated from the blood of 10 blood donors and 10 patients with CLL according to Boyum (1968). Tho isolated cells were homogenized in 0.1 % Triton X-100 in a glass homogenizer with a teflon pestle. The 105,000 x g x 30 min supernatants, dialyzed against 0.003 M NaCl were used for polyacxylamide gel electrophoresis. The 7.5 % gells were prepared according to Clarke (1964); pH of the electrode buffer was 8.5.
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Six 200 p1 samples of dialyzed supernatants containing 200-400 pg of protein (as determined by the method of Lowry et a1 (1951) were run simultaneously on 6 gels. After electrophoresis gels were stained for the detection of the activities of acid phosphatase (Lawrence et a1 1960, Melnick 1968), P-glucuronidase and a-naphthyl acetate esterase (Melnick 1968).
RESULTS
lymphocyte phosphatase were very similar. p-glucuronidase activity is represented by 2 cathodal bands in both normal and CLL lymphocytes as is shown in Figure 2. The quantitative differences of p-glucuronidase activity noted in the gels may be due to different protein content in the tested samples. In Figure 3 the isoenzymic patterns of a-naphthyl acetate esterase from normal
Figure 1 shows the isoenzyme patterns of acid phosphatase from leukaemic lymphocyte extract visualized by the use of 4 different substrates. The slowest (cathodal) band represents the main part of the activity and is detectable with all 4 substrates used. However, this band was hardly discernible when a-glycerophosphate was used as substrate. In the central part of the gels there were 2 bands of very low phosphatase activity. The patterns of CLL and normal
Figure 2. Disc gel electrophorograms of p-glucuronidase in the extracts of normal (left) and leukaemic lymphocytes (right).
Figure 1. Disc gel electrophorograms of acid phosphatases from the extracts of leukaemic lymphocytes with the use of following substrates: a-naphthyl-phosphate (A), naphthol AS-BI phosphate (B), p-glycero-phosphate (C) and a-glycero-phosphate (D).
Figure 3. Disc gel electrophorograms of cr-naphthy1 acetate esterase in the extracts of normal (left) and leukaemic lymphocytes (right).
HETEROGENEITY OF LYMPHOCYTIC HYDROLASES
and CLL lymphocytes are compared. One very strong cathodal band and 3 to 5 bands of low activity were found in the lymphocytes isolated from normal subjects. In contrast, CLL lymphocytes showed 2 very strong anodal bands of the esterase activity. Cathodal bands in CLL lymphocytes were either very weak or absent. DISCUSSION
The 3 tested enzymes in both normal and CLL lymphocytes are heterogeneous. Acid phosphatase activity is represented by one prominent cathodal band, which probably corresponds to band 3 in terms of Li et al nomenclature (Li et a1 1970a/b), and 2 very weak bands in the middle of the gel. It is not clear whether these bands are of lymphocytic origin, or come from contaminating cells - polymorphs or eosinophils, where they represent the bulk of activity (Pajdak & Lisiewicz 1973). It should be stressed, however, that the percentage of contaminating cells in our preparations of lymphocytes was less than 5 %. The activities of acid phosphatase and p-glucuronidase have previously. been reported to increase in patients with acute lymphoblastic leukaemia (Sippel et al 1975) and to decrease in CLL lymphocytes (Yam & Mitus 1968, Douglas et a1 1973). In spite of substantial quantitative differences, the isoenzymograms of these enzymes are similar in normal and CLL cells. The isoenzymic patterns of nonspecific esterase of normal and CLL lymphocytes exhibit striking differences. It should be kept in mind, however, that a-naphthyl acetate is readily hydrolyzed by several different proteolytic and lipoytic enzymes (Young & Bittar 1973, Li et a1 1973) and that nonspecific esterase is not exclusively of lysoScand J Haematol (1977) 19
209
soma1 origin (Mueller et al 1975). Nevertheless, the two prominent anodal bands found in CLL lymphocytes which are virtually absent from normal lymphocytes might originate from ‘leukaemic clone’. This suggestion may be to some degree consistent with the findings of Barr & Perry (1976) indicating that all T cells show a granular positivity for the presence of nonspecific eaterase, while noa T cells uniformly give a diffuse cytoplasmic stain. CLL is generally believed to be a B cell connected proliferative disease (Preud’homme & Seligman 1972, Rowlands et a1 1974). Furthermore, according to T h m s o n & Robinson (1967) in the blood of patients with CLL there is a definite part of colchicine ultrasensitive lymphocytes, which is suggested to represent ‘pathological‘ subpopulation, coexisting with colchicine nm-ultrasensitive ‘normal’ lymphocyte subpopulation. Work is now in progress on esterase isoenzymes of these 2 subpopulatims, which may allow a better understanding of this problem. ACKNOWLEDGEMENTS The authors thank Miss Graiyna Cierpikowska for skilled technical assistance. This work was supported in part by the Polish Academy of Sciences - PAN 337NI.
REFERENCES Allison A C (1969) Lysosomes and cancer. In J T Dingle & H B Fell (eds) Lysosomes in biology and pathology. North-Holland Publishing, Amsterdam. Barr R D & Perry S (1976) Lysmomal acid hydrolases in human lymphocyte subpopulations. B r J Haematol 32, 565-72. Boyum A (1968) Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest, Suppl 97. 14
210
A. CELINSKI ’& W. PAJDAK
Clarke T J (1964) Simplified ‘disc’ (polyacrylamide gel) electrophoresis. Ann N Y Acad Sci 121, 428-36. Douglas S D, Cohnen G, Konig E & Brittinger G (1973) Lymphocyte lysosomes and lysosomal enzymes in chronic lymphocytic leukemia. Blood 41, 511-18. Lawrence S H P, Melnick P J & Weiner H E (1960) A species comparison of serum proteins and enzyme by starch electrophoresis. Proc Soc Exptl Biol Med 105, 572-75. Li C Y, Lam K W & Yam L T (1970a) Acid phosphatase isoenzymes in human leukocytes in normal and pathologic conditions. J Histochem Cytochem 18, 473-81. Li C Y, Lam K W & Yam L T (1970b) Studies of acid phosphatase isoenzymes in human leukocytes - demonstration of isoenzyme cell specificity. J Histochern Cytochem 18, 901-10. Li C Y, Lam K W & Yam L T (1973) Esterases in human leukocytes. J Histochem Cytochem 21, 1-12. Lowry 0 K, Rmenbrough N J, Farr A L & Randall R J (1951) Pratein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75. Melnick P J (1968) Histochemical enzymology of leukemic cells, Chapter 4. In G D Amromin (ed) Pathology of leukemia. New York. Mueller I, Brun del R e G, Buerki H, Keller H U, Hess M W & Cottier H (1975) Nonspecific acid esterase activity: a criterion for differentiation
of T and B lymphocytes in mouse lymph nodes. Eur J Immunol 5, 270-74. Pajdak W & Lisiewicz J (1973) Heterogeneity of enzymes in leukaemic eosinophils. Scand J Haematol 11, 325-31. Preud‘homme J L & Seligmann M (1972) Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40, 777-94. Rowlands D T Jr, Daniele R P, Nowell P C & Wurzel H A (1974) Characterization of lymphocyte subpopulations in chronic lymphocytic leukemia. Cancer 34, 1962-70. Sippel W G , Antonowicz S, Lazarus H & Schwachman H (1975) Lysosomal and mitochondria1 enzyme activities in human lymphoid cell lines obtained from children with acute lymphoblastic leukemia and controls. Exptl Cell Res 91, 15258. Thomson A E R & Robinson M A (1967) Cytocidal action of colchicine in vitro on lymphocytes in chronic lymphocytic leukemia. Lancet 2, 868-70. Yam L T & Mitus T J (1968) The lymphocyte beta-glucuronidase activity in lymphoproliferative disorders. Blood 31, 48CL89. Young C W & Bittar E S (1973) Analysis of tissue esterases from patients with Hodgkin’s disease and other types of advanced cancer by isoelectric focusing in acrylamide gel. Cancer Res 33, 2692-2700.
Heterogeneity of Acid Phosphatase,
p - Glucuronidase, and a -Naphthyl-Acetate Esterase in Normal and Leukaemic Lymphocytes Aleksander Celidski #&Wladyslaw Pajdak Department of Clinical Chemistry, Institute of Internal Medicine, Medical Academy Cracow, Poland
Comparative study of isocnzymic patterns of acid phosphatase, P-glucuronidase and a-naphthyl acetate esterase of normal and chronic lymphocytic leukaemia (CLL) lymphocytes was carried out by means of polyacrylamide gel electrophoresis. The isoenzymograms of acid phosphatase and P-glucuronidase of leukaemic cells were similar to that of normal lymphocytes. However, different patterns were found in the case of a-naphthyl acetate esterase. Two prominent anodal bands were present in CLL lymphocytes; these bands were either absent or very weak in normal cells. Key words: chr. lymphocytic leukaemia - isoenzymes - lysosyme hydrolase polyacrylamide gel electrophoresis
-
Accepted for publication April 18, 1977 Correspondence to: Dr. Aleksander Celihski, Department o'f Clinical Chemistry, Institute of Internal Medicine, Kopernika 17, 31-501 Krakow, Poland
Despite many comparative cytochemical studies on normal and leukaemic lymphocytes, and extensive studies on blastic transformation in culture of both normal and CLL cells, information concerning the isoenzymic pattems of lymphocytic enzymes is scanty. It is not excluded that CLL may be caused by disturbances of lysosomal enzymes (Allison 1969) on the other hand, the disease may cause changes in activities of these enzymes (Douglas et al 1973). Hence, information on the shifts in isoenzymograms seems to be of importance for better understanding of the leukaemic process.
The purpose of current repo'rt is to compare the isoenzymic patterns of 3 enzymes of nolrmal and CLL lymphocytes. MATERIALS AND METHODS Normal and CLL lymphocytes were isolated from the blood of 10 blood donors and 10 patients with CLL according to Boyum (1968). Tho isolated cells were homogenized in 0.1 % Triton X-100 in a glass homogenizer with a teflon pestle. The 105,000 x g x 30 min supernatants, dialyzed against 0.003 M NaCl were used for polyacxylamide gel electrophoresis. The 7.5 % gells were prepared according to Clarke (1964); pH of the electrode buffer was 8.5.
A. CELINSKI & W. PAJDAK
208
Six 200 p1 samples of dialyzed supernatants containing 200-400 pg of protein (as determined by the method of Lowry et a1 (1951) were run simultaneously on 6 gels. After electrophoresis gels were stained for the detection of the activities of acid phosphatase (Lawrence et a1 1960, Melnick 1968), P-glucuronidase and a-naphthyl acetate esterase (Melnick 1968).
RESULTS
lymphocyte phosphatase were very similar. p-glucuronidase activity is represented by 2 cathodal bands in both normal and CLL lymphocytes as is shown in Figure 2. The quantitative differences of p-glucuronidase activity noted in the gels may be due to different protein content in the tested samples. In Figure 3 the isoenzymic patterns of a-naphthyl acetate esterase from normal
Figure 1 shows the isoenzyme patterns of acid phosphatase from leukaemic lymphocyte extract visualized by the use of 4 different substrates. The slowest (cathodal) band represents the main part of the activity and is detectable with all 4 substrates used. However, this band was hardly discernible when a-glycerophosphate was used as substrate. In the central part of the gels there were 2 bands of very low phosphatase activity. The patterns of CLL and normal
Figure 2. Disc gel electrophorograms of p-glucuronidase in the extracts of normal (left) and leukaemic lymphocytes (right).
Figure 1. Disc gel electrophorograms of acid phosphatases from the extracts of leukaemic lymphocytes with the use of following substrates: a-naphthyl-phosphate (A), naphthol AS-BI phosphate (B), p-glycero-phosphate (C) and a-glycero-phosphate (D).
Figure 3. Disc gel electrophorograms of cr-naphthy1 acetate esterase in the extracts of normal (left) and leukaemic lymphocytes (right).
HETEROGENEITY OF LYMPHOCYTIC HYDROLASES
and CLL lymphocytes are compared. One very strong cathodal band and 3 to 5 bands of low activity were found in the lymphocytes isolated from normal subjects. In contrast, CLL lymphocytes showed 2 very strong anodal bands of the esterase activity. Cathodal bands in CLL lymphocytes were either very weak or absent. DISCUSSION
The 3 tested enzymes in both normal and CLL lymphocytes are heterogeneous. Acid phosphatase activity is represented by one prominent cathodal band, which probably corresponds to band 3 in terms of Li et al nomenclature (Li et a1 1970a/b), and 2 very weak bands in the middle of the gel. It is not clear whether these bands are of lymphocytic origin, or come from contaminating cells - polymorphs or eosinophils, where they represent the bulk of activity (Pajdak & Lisiewicz 1973). It should be stressed, however, that the percentage of contaminating cells in our preparations of lymphocytes was less than 5 %. The activities of acid phosphatase and p-glucuronidase have previously. been reported to increase in patients with acute lymphoblastic leukaemia (Sippel et al 1975) and to decrease in CLL lymphocytes (Yam & Mitus 1968, Douglas et a1 1973). In spite of substantial quantitative differences, the isoenzymograms of these enzymes are similar in normal and CLL cells. The isoenzymic patterns of nonspecific esterase of normal and CLL lymphocytes exhibit striking differences. It should be kept in mind, however, that a-naphthyl acetate is readily hydrolyzed by several different proteolytic and lipoytic enzymes (Young & Bittar 1973, Li et a1 1973) and that nonspecific esterase is not exclusively of lysoScand J Haematol (1977) 19
209
soma1 origin (Mueller et al 1975). Nevertheless, the two prominent anodal bands found in CLL lymphocytes which are virtually absent from normal lymphocytes might originate from ‘leukaemic clone’. This suggestion may be to some degree consistent with the findings of Barr & Perry (1976) indicating that all T cells show a granular positivity for the presence of nonspecific eaterase, while noa T cells uniformly give a diffuse cytoplasmic stain. CLL is generally believed to be a B cell connected proliferative disease (Preud’homme & Seligman 1972, Rowlands et a1 1974). Furthermore, according to T h m s o n & Robinson (1967) in the blood of patients with CLL there is a definite part of colchicine ultrasensitive lymphocytes, which is suggested to represent ‘pathological‘ subpopulation, coexisting with colchicine nm-ultrasensitive ‘normal’ lymphocyte subpopulation. Work is now in progress on esterase isoenzymes of these 2 subpopulatims, which may allow a better understanding of this problem. ACKNOWLEDGEMENTS The authors thank Miss Graiyna Cierpikowska for skilled technical assistance. This work was supported in part by the Polish Academy of Sciences - PAN 337NI.
REFERENCES Allison A C (1969) Lysosomes and cancer. In J T Dingle & H B Fell (eds) Lysosomes in biology and pathology. North-Holland Publishing, Amsterdam. Barr R D & Perry S (1976) Lysmomal acid hydrolases in human lymphocyte subpopulations. B r J Haematol 32, 565-72. Boyum A (1968) Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest, Suppl 97. 14
210
A. CELINSKI ’& W. PAJDAK
Clarke T J (1964) Simplified ‘disc’ (polyacrylamide gel) electrophoresis. Ann N Y Acad Sci 121, 428-36. Douglas S D, Cohnen G, Konig E & Brittinger G (1973) Lymphocyte lysosomes and lysosomal enzymes in chronic lymphocytic leukemia. Blood 41, 511-18. Lawrence S H P, Melnick P J & Weiner H E (1960) A species comparison of serum proteins and enzyme by starch electrophoresis. Proc Soc Exptl Biol Med 105, 572-75. Li C Y, Lam K W & Yam L T (1970a) Acid phosphatase isoenzymes in human leukocytes in normal and pathologic conditions. J Histochem Cytochem 18, 473-81. Li C Y, Lam K W & Yam L T (1970b) Studies of acid phosphatase isoenzymes in human leukocytes - demonstration of isoenzyme cell specificity. J Histochern Cytochem 18, 901-10. Li C Y, Lam K W & Yam L T (1973) Esterases in human leukocytes. J Histochem Cytochem 21, 1-12. Lowry 0 K, Rmenbrough N J, Farr A L & Randall R J (1951) Pratein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75. Melnick P J (1968) Histochemical enzymology of leukemic cells, Chapter 4. In G D Amromin (ed) Pathology of leukemia. New York. Mueller I, Brun del R e G, Buerki H, Keller H U, Hess M W & Cottier H (1975) Nonspecific acid esterase activity: a criterion for differentiation
of T and B lymphocytes in mouse lymph nodes. Eur J Immunol 5, 270-74. Pajdak W & Lisiewicz J (1973) Heterogeneity of enzymes in leukaemic eosinophils. Scand J Haematol 11, 325-31. Preud‘homme J L & Seligmann M (1972) Surface bound immunoglobulins as a cell marker in human lymphoproliferative diseases. Blood 40, 777-94. Rowlands D T Jr, Daniele R P, Nowell P C & Wurzel H A (1974) Characterization of lymphocyte subpopulations in chronic lymphocytic leukemia. Cancer 34, 1962-70. Sippel W G , Antonowicz S, Lazarus H & Schwachman H (1975) Lysosomal and mitochondria1 enzyme activities in human lymphoid cell lines obtained from children with acute lymphoblastic leukemia and controls. Exptl Cell Res 91, 15258. Thomson A E R & Robinson M A (1967) Cytocidal action of colchicine in vitro on lymphocytes in chronic lymphocytic leukemia. Lancet 2, 868-70. Yam L T & Mitus T J (1968) The lymphocyte beta-glucuronidase activity in lymphoproliferative disorders. Blood 31, 48CL89. Young C W & Bittar E S (1973) Analysis of tissue esterases from patients with Hodgkin’s disease and other types of advanced cancer by isoelectric focusing in acrylamide gel. Cancer Res 33, 2692-2700.
