American Journal of Hematology 6:381-386 (1979)
Acid Phosphatase in Leukemic Blasts: Characterization by lsoelectric Focusing in Polyacrylamide Gel Lawrence Kass and Donald Munster Department of internal Medicine (Simpson Memorial institute), The University of Michigan, Ann Arbor
Using the high resolution technique of isoelectric focusing in polyacrylamide gel, isoenzymatic components of acid phosphatase were detected in cell-free extracts prepared from different cytologic types of leukemic blasts in adults. Results indicate that for different cytologic types, different characteristic patterns of acid phosphatase isoenzyme could be detected. These studies extend conventional cytochemistry and indicate that characteristic patterns of acid phosphatase isoenzyme can be detected for various cytologic types of acute leukemia. Key words: acid phosphatase, leukemia, isoenzymes, electrophoresis
I NTRODUCT 10N
Acid phosphatase is a lysosomal enzyme found in a wide variety of hematopoietic cells [ l , 51. Cytochemically, acid phosphatase activity can be detected with the use of a-naphthol phosphate as substrate and one of several azo-dye indicators [ 1, 51 . Cytochemical detection of acid phosphatase has been utilized as a diagnostic tool. For example, erythroblasts in erythroleukemia contain a unique unipolar localization of enzymatic activity [4] . Similar unipolar localization of acid phosphatase activity occurs in T-cell leukemic lymphoblasts of the convoluted type [ 6 ] . In hairy cells from patients with hairy cell leukemia, acid phosphatase activity is resistant t o inhibition by L-tartrate [8]. This enzymatic property may be caused by the presence of a distinctive acid phosphatase isoenzyme found only in hairy cells [ 8 ] Advances in electrophoretic techniques have extended applications of cytoSupported by grants from the National Leukemia Association and the Children’s Leukemia Foundation of Michigan. Received for publication August 15, 1978; accepted April 4, 1979. Address reprint requests to Lawrence Kass, MD, Department of Internal Medicine (Simpson Memorial Institute), The University of Michigan, Ann Arbor, MI 48109.
0361-8609/79/0604-0381$01.40 0 1979 Alan R. Liss, Inc.
382
Kass and Munster
chemistry. Using conventional electrophoretic methods, isoenzymatic analysis of acid phosphatase extracted from normal and pathologic blood cells has been reported [5] . Similarly, isoenzymatic analyses of nonspecific esterases extracted from leukemic blasts in adult acute leukemia have been described [2] . In these studies, different isoenzymatic patterns seemed to correlate with specific cell types. However, results and their subsequent interpretation were limited by the electrophoretic methods available at the time. For example, many electrophoretic gels contained diffusely stained areas, suggesting that there may have been additional unresolved components within them. The newly-developed technique of isoelectric focusing in polyacrylamide gel has recently been applied to enzymes. This technique makes it possible t o detect isoenzymatic components that could not be resolved by using conventional electrophoretic methods. With isoelectric focusing, numerous isoenzymatic components have been detected in nonspecific esterases extracted from tissues of patients with Hodgkin disease [9, 101 as well as from a variety of cytologic types of leukemic blasts from adults with acute leukemia [3]. Because of the importance of acid phosphatase in cytochemistry and the increasing applications of isoelectric focusing of enzymes, we studied isoenzymatic components of acid phosphatase in extracts from a wide variety of cytologic types of leukemic blasts. In these studies, we also compared isoenzymatic patterns of acid phosphatase in leukemic blasts to those found in acid phosphatase from normal human blood cells, Results suggest that for each cytologic type there may be distinctive isoenzymatic patterns of acid phosphatase. These patterns differ from those found in normal cellular counterparts. MATERIALS AND METHODS
At the time of diagnosis and prior to any treatment, leukemic blasts were outained from peripheral venous blood of five patients with acute lymphoblastic leukemia, from three patients with acute myeloblastic leukemia, and four patients with acute monocytic leukemia. For cytochemical studies, films of leukemic blasts were stained for glycogen using the PAS (periodic acid-Schiff) reagent [ 1] and for specific esterase activity using naphthol ASD-chloroacetate as substrate [4] . Other stains included nonspecific esterase activity using a-naphthyl acetate as substrate [4] with fluoride inhibition, and acid phosphatase [ l ] . Cytologic and cytochemical identification of leukemic blasts was achieved with the use of currently accepted criteria [ 1 , 7 ] and all of the cases were regarded as typical. Leukemic blasts were isolated from blood samples by the methods described previously [2] and they contained virtually no erythrocytes or platelets. Also, the leukemic cell populations were homogeneous. Highly-purified preparations of lymphocytes, granulocytes, and monocytes were obtained from the peripheral blood of five presumably normal persons for the purpose of comparison and control [3]. For each electrophoretic gel, approximately 6 X 10" cells were used. Cells were disrupted by sonication [2] and cell-free supernatants were applied t o polyacrylamide gel containing ampholyte carriers (ampholine, LKB Laboratories, Stockholm) for isoelectric focusing [9, l o ] . Acid phosphatase activity was demonstrated using a-naphthol phosphate as sub-
Acid Phosphatase in Leukemic Blasts
383
strate and Fast Garnet GBC as dye coupler [ 3 ] .Separate identical gels were incubated in the presence of 50 mM L-tartrate for inhibition studies [8]. Using a -naphthol phosphate and Fast Garnet GBC, the color reaction indicating enzymatic activity developed rapidly and faded quickly. Accordingly, as soon as possible after incubation and at the point of maximal color development of the dye-indicator, gels were photographed with Kodak high contrast copy film to avoid diffusion of isoenzymatic components. RESULTS
Figure 1 illustrates isoenzymatic patterns of acid phosphatase from highlypurified preparations of normal lymphocytes, granulocytes, and monocytes. Acid phosphatase from lymphocytes showed 7 isoenzymatic components, granulocytes demonstrated 13-14 components, and monocytes contained 4-5 components. With inclusion of tartrate in the incubation medium, granulocytes demonstrated one cathodal (fast-moving) band. Lymphocytes and monocytes revealed no resistant bands. Figure 2 depicts isoenzymatic patterns of acid phosphatase from leukemic blasts of patients with acute lymphoblastic leukemia, acute myeloblastic leukemia, and acute monocytic leukemia. Acid phosphatase from lymphoblasts contained 12 components. The slowest moving bands (anodal) showed the greatest sensitivity to tartrate inhibition; in addition, the anodal bands were relatively resistant t o tartrate. Acid phosphatase from myeloblasts showed 6-7 components. Of these, only the most anodal and cathodal bands were tartrate-resistant. Acid phosphatase from leukemic monocytes contained 10 anodal components and all of them were markedly sensitive to inhibition by tartrate. In comparing isoenzymatic patterns of acid phosphatase from leukemic blasts, cathodal components were detected only in enzyme from lymphoblasts and myeloblasts. In lymphoblasts and myeloblasts, anodal components showed similar electrophoretic patterns, except that there were more components in lymphoblasts than in myeloblasts. Acid phosphatase from leukemic monocytes showed more isoenzymatic components than enzyme from either lymphoblasts or myeloblasts. In monocytes, numerous isoenzymatic components were detected and all of them were anodal. Of the various isoenzymatic components, the most anodal of the large group of bands near the middle of each gel occurred in acid phosphatases from all cell types. Insofar as the other isoenzymatic components were concerned, no consistent similarities could be noted among the cell types. Aside from minor variations in the intensity of some of the bands, these patterns were consistent among the various cytologic types of leukemic blasts. In comparing the acid phosphatase from leukemic blasts t o the enzyme obtained from their normal cellular counterparts, more isoenzymatic components were found in leukemic lymphoblasts and monocytes than in normal lymphocytes or monocytes. Acquisition of new isoenzymatic components was particularly notable in acid phosphatase from leukemic monocytes compared to acid phosphatase from normal monocytes. Myeloblasts contained fewer isoenzymatic components than did preparations of mature granulocytes, perhaps reflecting immaturity of myeloblasts.
384
Kass and Munster
Fig. 1 . Isoelectric focusing of acid phosphatase activity in normal blood cells. Adjacent to the set of gels is a schematic representation of the electrophoretic bands: A. Lymphocytes; B. Granulocytes; C. Monocytes.
DISCUSSION
Isoenzymatic patterns of acid phosphatase obtained from leukemic lymphoblasts and leukemic monocytes showed more isoenzymatic components than their normal cellular counterparts. These findings suggest that although some leukemic blasts are “primitive” morphologically, they may be capable of synthesizing a larger number of isoenzymatic components of acid phosphatase than normal blood cells of the same or similar cytologic type. Normal mature granulocytes contained more isoenzymatic components than leukemic myeloblasts. These findings suggest that in
Acid Phosphatase in Leukemic Blasts
Fig. 2. Isoelectric focusing of acid phosphatase in various cytologic types of leukemic blasts. Adjacent to the set of gels is a schematic representation of the electrophoretic bands: A. Acute lymphoblastic leukemia; B. Acute lymphoblastic leukemia with tartrate; C. Acute myeloblastic leukemia; D. Acute myeloblastic leukemia with tartrate; E. Acute monocytic leukemia; F. Acute monocytic leukemia with tartrate.
granulocytic cells, elaboration of isoenzymatic components may be a function of cellular maturation. Future studies utilizing preparations of normal myeloblasts may help to resolve the question of whether isoenzymatic patterns are related to the leukemic nature of the cells or whether they represent a normal feature of immature cells. Combined with conventional cytochemical techniques, isoelectric focusing of acid phosphatase and other enzymes may amplify and refine the cytochemical and morphologic criteria currently available for leukemic blasts.
385
386
Kass and Munster
REFERENCES 1. Hayhoe FGJ, Quaglino D, Doll R: “Cytology and Cytochemistry of Acute Leukemia.” London: H.M. Stationery Office, 1964. 2. Kass L, Peters CL: Esterases in acute leukemia: A cytochemical and electrophoretic study. Am J Clin Pathol 69:57, 1978. 3. Kass L, Munster D: Nonspecific esterases in leukemic blasts: Characterization by isoelectric focusing in polyacrylamide gel. Am J Clin Pathol (In press). 4. Leder LD: Die fermentcytochemische Erkennung normaler und neoplastischer Erythropoiesezellen im Schnitt und Ausstrich. Blut 16:86, 1967. 5. Li CY, Yam LT, Lam KW: Acid phosphatase isoenzyme in human leukocytes in normal and pathological conditions. J Histochem Cytochem 18:473, 1970. 6. Stein H et al: Lymphoblastic lymphoma of convoluted or acid phosphatase type - A tumor of T-precursor cells. Int J Cancer 17:292, 1976. 7. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol55:283, 1971. 8. Yam LT, Li CY, Lam KW: Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. N Engl J Med 284:357, 1971. 9. Young CW,Bittar ES: Analysis of tissue esterases from patients with Hodgkin disease and other types of advanced cancer by isoelectric focusing in acrylamide gel. Can Res 33: 2692, 1973. 10. Young CW, Bittar ES: Isoelectric focusing comparison of human tissue esterases with those from normal and Bacillus Calmette-Guerin treated mice. Can Res 34:2675, 1974.
Acid Phosphatase in Leukemic Blasts: Characterization by lsoelectric Focusing in Polyacrylamide Gel Lawrence Kass and Donald Munster Department of internal Medicine (Simpson Memorial institute), The University of Michigan, Ann Arbor
Using the high resolution technique of isoelectric focusing in polyacrylamide gel, isoenzymatic components of acid phosphatase were detected in cell-free extracts prepared from different cytologic types of leukemic blasts in adults. Results indicate that for different cytologic types, different characteristic patterns of acid phosphatase isoenzyme could be detected. These studies extend conventional cytochemistry and indicate that characteristic patterns of acid phosphatase isoenzyme can be detected for various cytologic types of acute leukemia. Key words: acid phosphatase, leukemia, isoenzymes, electrophoresis
I NTRODUCT 10N
Acid phosphatase is a lysosomal enzyme found in a wide variety of hematopoietic cells [ l , 51. Cytochemically, acid phosphatase activity can be detected with the use of a-naphthol phosphate as substrate and one of several azo-dye indicators [ 1, 51 . Cytochemical detection of acid phosphatase has been utilized as a diagnostic tool. For example, erythroblasts in erythroleukemia contain a unique unipolar localization of enzymatic activity [4] . Similar unipolar localization of acid phosphatase activity occurs in T-cell leukemic lymphoblasts of the convoluted type [ 6 ] . In hairy cells from patients with hairy cell leukemia, acid phosphatase activity is resistant t o inhibition by L-tartrate [8]. This enzymatic property may be caused by the presence of a distinctive acid phosphatase isoenzyme found only in hairy cells [ 8 ] Advances in electrophoretic techniques have extended applications of cytoSupported by grants from the National Leukemia Association and the Children’s Leukemia Foundation of Michigan. Received for publication August 15, 1978; accepted April 4, 1979. Address reprint requests to Lawrence Kass, MD, Department of Internal Medicine (Simpson Memorial Institute), The University of Michigan, Ann Arbor, MI 48109.
0361-8609/79/0604-0381$01.40 0 1979 Alan R. Liss, Inc.
382
Kass and Munster
chemistry. Using conventional electrophoretic methods, isoenzymatic analysis of acid phosphatase extracted from normal and pathologic blood cells has been reported [5] . Similarly, isoenzymatic analyses of nonspecific esterases extracted from leukemic blasts in adult acute leukemia have been described [2] . In these studies, different isoenzymatic patterns seemed to correlate with specific cell types. However, results and their subsequent interpretation were limited by the electrophoretic methods available at the time. For example, many electrophoretic gels contained diffusely stained areas, suggesting that there may have been additional unresolved components within them. The newly-developed technique of isoelectric focusing in polyacrylamide gel has recently been applied to enzymes. This technique makes it possible t o detect isoenzymatic components that could not be resolved by using conventional electrophoretic methods. With isoelectric focusing, numerous isoenzymatic components have been detected in nonspecific esterases extracted from tissues of patients with Hodgkin disease [9, 101 as well as from a variety of cytologic types of leukemic blasts from adults with acute leukemia [3]. Because of the importance of acid phosphatase in cytochemistry and the increasing applications of isoelectric focusing of enzymes, we studied isoenzymatic components of acid phosphatase in extracts from a wide variety of cytologic types of leukemic blasts. In these studies, we also compared isoenzymatic patterns of acid phosphatase in leukemic blasts to those found in acid phosphatase from normal human blood cells, Results suggest that for each cytologic type there may be distinctive isoenzymatic patterns of acid phosphatase. These patterns differ from those found in normal cellular counterparts. MATERIALS AND METHODS
At the time of diagnosis and prior to any treatment, leukemic blasts were outained from peripheral venous blood of five patients with acute lymphoblastic leukemia, from three patients with acute myeloblastic leukemia, and four patients with acute monocytic leukemia. For cytochemical studies, films of leukemic blasts were stained for glycogen using the PAS (periodic acid-Schiff) reagent [ 1] and for specific esterase activity using naphthol ASD-chloroacetate as substrate [4] . Other stains included nonspecific esterase activity using a-naphthyl acetate as substrate [4] with fluoride inhibition, and acid phosphatase [ l ] . Cytologic and cytochemical identification of leukemic blasts was achieved with the use of currently accepted criteria [ 1 , 7 ] and all of the cases were regarded as typical. Leukemic blasts were isolated from blood samples by the methods described previously [2] and they contained virtually no erythrocytes or platelets. Also, the leukemic cell populations were homogeneous. Highly-purified preparations of lymphocytes, granulocytes, and monocytes were obtained from the peripheral blood of five presumably normal persons for the purpose of comparison and control [3]. For each electrophoretic gel, approximately 6 X 10" cells were used. Cells were disrupted by sonication [2] and cell-free supernatants were applied t o polyacrylamide gel containing ampholyte carriers (ampholine, LKB Laboratories, Stockholm) for isoelectric focusing [9, l o ] . Acid phosphatase activity was demonstrated using a-naphthol phosphate as sub-
Acid Phosphatase in Leukemic Blasts
383
strate and Fast Garnet GBC as dye coupler [ 3 ] .Separate identical gels were incubated in the presence of 50 mM L-tartrate for inhibition studies [8]. Using a -naphthol phosphate and Fast Garnet GBC, the color reaction indicating enzymatic activity developed rapidly and faded quickly. Accordingly, as soon as possible after incubation and at the point of maximal color development of the dye-indicator, gels were photographed with Kodak high contrast copy film to avoid diffusion of isoenzymatic components. RESULTS
Figure 1 illustrates isoenzymatic patterns of acid phosphatase from highlypurified preparations of normal lymphocytes, granulocytes, and monocytes. Acid phosphatase from lymphocytes showed 7 isoenzymatic components, granulocytes demonstrated 13-14 components, and monocytes contained 4-5 components. With inclusion of tartrate in the incubation medium, granulocytes demonstrated one cathodal (fast-moving) band. Lymphocytes and monocytes revealed no resistant bands. Figure 2 depicts isoenzymatic patterns of acid phosphatase from leukemic blasts of patients with acute lymphoblastic leukemia, acute myeloblastic leukemia, and acute monocytic leukemia. Acid phosphatase from lymphoblasts contained 12 components. The slowest moving bands (anodal) showed the greatest sensitivity to tartrate inhibition; in addition, the anodal bands were relatively resistant t o tartrate. Acid phosphatase from myeloblasts showed 6-7 components. Of these, only the most anodal and cathodal bands were tartrate-resistant. Acid phosphatase from leukemic monocytes contained 10 anodal components and all of them were markedly sensitive to inhibition by tartrate. In comparing isoenzymatic patterns of acid phosphatase from leukemic blasts, cathodal components were detected only in enzyme from lymphoblasts and myeloblasts. In lymphoblasts and myeloblasts, anodal components showed similar electrophoretic patterns, except that there were more components in lymphoblasts than in myeloblasts. Acid phosphatase from leukemic monocytes showed more isoenzymatic components than enzyme from either lymphoblasts or myeloblasts. In monocytes, numerous isoenzymatic components were detected and all of them were anodal. Of the various isoenzymatic components, the most anodal of the large group of bands near the middle of each gel occurred in acid phosphatases from all cell types. Insofar as the other isoenzymatic components were concerned, no consistent similarities could be noted among the cell types. Aside from minor variations in the intensity of some of the bands, these patterns were consistent among the various cytologic types of leukemic blasts. In comparing the acid phosphatase from leukemic blasts t o the enzyme obtained from their normal cellular counterparts, more isoenzymatic components were found in leukemic lymphoblasts and monocytes than in normal lymphocytes or monocytes. Acquisition of new isoenzymatic components was particularly notable in acid phosphatase from leukemic monocytes compared to acid phosphatase from normal monocytes. Myeloblasts contained fewer isoenzymatic components than did preparations of mature granulocytes, perhaps reflecting immaturity of myeloblasts.
384
Kass and Munster
Fig. 1 . Isoelectric focusing of acid phosphatase activity in normal blood cells. Adjacent to the set of gels is a schematic representation of the electrophoretic bands: A. Lymphocytes; B. Granulocytes; C. Monocytes.
DISCUSSION
Isoenzymatic patterns of acid phosphatase obtained from leukemic lymphoblasts and leukemic monocytes showed more isoenzymatic components than their normal cellular counterparts. These findings suggest that although some leukemic blasts are “primitive” morphologically, they may be capable of synthesizing a larger number of isoenzymatic components of acid phosphatase than normal blood cells of the same or similar cytologic type. Normal mature granulocytes contained more isoenzymatic components than leukemic myeloblasts. These findings suggest that in
Acid Phosphatase in Leukemic Blasts
Fig. 2. Isoelectric focusing of acid phosphatase in various cytologic types of leukemic blasts. Adjacent to the set of gels is a schematic representation of the electrophoretic bands: A. Acute lymphoblastic leukemia; B. Acute lymphoblastic leukemia with tartrate; C. Acute myeloblastic leukemia; D. Acute myeloblastic leukemia with tartrate; E. Acute monocytic leukemia; F. Acute monocytic leukemia with tartrate.
granulocytic cells, elaboration of isoenzymatic components may be a function of cellular maturation. Future studies utilizing preparations of normal myeloblasts may help to resolve the question of whether isoenzymatic patterns are related to the leukemic nature of the cells or whether they represent a normal feature of immature cells. Combined with conventional cytochemical techniques, isoelectric focusing of acid phosphatase and other enzymes may amplify and refine the cytochemical and morphologic criteria currently available for leukemic blasts.
385
386
Kass and Munster
REFERENCES 1. Hayhoe FGJ, Quaglino D, Doll R: “Cytology and Cytochemistry of Acute Leukemia.” London: H.M. Stationery Office, 1964. 2. Kass L, Peters CL: Esterases in acute leukemia: A cytochemical and electrophoretic study. Am J Clin Pathol 69:57, 1978. 3. Kass L, Munster D: Nonspecific esterases in leukemic blasts: Characterization by isoelectric focusing in polyacrylamide gel. Am J Clin Pathol (In press). 4. Leder LD: Die fermentcytochemische Erkennung normaler und neoplastischer Erythropoiesezellen im Schnitt und Ausstrich. Blut 16:86, 1967. 5. Li CY, Yam LT, Lam KW: Acid phosphatase isoenzyme in human leukocytes in normal and pathological conditions. J Histochem Cytochem 18:473, 1970. 6. Stein H et al: Lymphoblastic lymphoma of convoluted or acid phosphatase type - A tumor of T-precursor cells. Int J Cancer 17:292, 1976. 7. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol55:283, 1971. 8. Yam LT, Li CY, Lam KW: Tartrate-resistant acid phosphatase isoenzyme in the reticulum cells of leukemic reticuloendotheliosis. N Engl J Med 284:357, 1971. 9. Young CW,Bittar ES: Analysis of tissue esterases from patients with Hodgkin disease and other types of advanced cancer by isoelectric focusing in acrylamide gel. Can Res 33: 2692, 1973. 10. Young CW, Bittar ES: Isoelectric focusing comparison of human tissue esterases with those from normal and Bacillus Calmette-Guerin treated mice. Can Res 34:2675, 1974.
