Conversion of skeletal tartrate-sensitive acid phosphatases into tartrate-resistant isoenzymes in vitro.

1. Chicken skeletal tartrate-sensitive (TsACP) and -resistant (TrACP) acid phosphatase isoenzymes could be separated from each other by carboxylmethyl-sepharose ion exchange chromatography. 2. Chicken skeletal TsACP showed a gradual time-dependent loss of sensitivity to tartrate inhibition when incubated at room temperature, but not at 4 degrees C. 3. The loss of sensitivity to tartrate inhibition was associated with an activation of the enzyme activity. 4. These changes were accompanied with a shift in the electrophoretic mobility of the enzyme activity from a large molecular sized form to a smaller molecular sized form that resembled the freshly prepared TrACP on the native acidic polyacrylamide electrophoresis gels, and on molecular sieve Superose-12 Fast Protein Liquid Chromatography. 5. Kinetic evaluations of the biochemical properties of the "converted" TsACP activity resembled the TrACP. 6. The apparent "conversion" was not unique to chicken TsACP, since similar "conversion" was observed with partially purified preparations of bovine bone matrix TsACP and of human osteoblastic TsACP. 7. Addition of several serine protease inhibitors did not prevent the "conversion". 8. These findings are consistent with the possibility that skeletal TsACPs are precursors of skeletal TrACPs.     

Characterization and purification of acid phosphatase from ancient human bone

In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10 degrees C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.     

Characterization and Purification of Acid Phosphatase from Ancient Human Bone

Abstract

In this research, acid phosphatase was purified and characterized from approximately 3000-year-old human bones from archeological excavations. Using anion exchange chromatography, two isoenzymes, TrACP and TsACP, were isolated from the bone. TrACP and TsACP were eluted separately, with a concentration gradient, from a CM-sepharose column. The resulting TrACP was further purified on a cellulose phosphate column. The activity was determined by using pNPP as substrate. Additionally, protein was determined by the Bradford and Coomassie Brilliant Blue method. The optimum pHs of TsACP and TrACP were 6 and 5, respectively. The optimum temperatures were 0 and 10°C, respectively. Molecular weights were measured by gel filtration chromatography. The isoenzyme purity was checked with SDS-PAGE. Finally, the effects of sodium molybdate and tartrate on isoenzyme activity were determined.     

Establishment and characterization of an Epstein-Barr virus nuclear antigen-negative hairy cell line

Continuous cell lines have been established from spleen cells of patients with confirmed hairy cell leukemia (HCL). One cell line, HCL-Z1, lacks Epstein-Barr virus nuclear antigen (EBNA), grows attached to the substratum and retains typical features of hairy cells as revealed by transmission and scanning electron microscopy. HCL-Z1 differs morphologically from the three other EBNA-positive lymphoblastoid cell lines obtained (HCL-Z2, HCL-Z3, HCL-Z4) as well as from normal spleen cells or lymphocytes. The three lymphoblastoid cell lines derived from HCL patients show similar surface features as a line from a myeloma patient. Therefore, not all cell lines derived from HCL patients may be considered as representative of the patients leukemia cells.     

Purification and N-terminal sequence of two tartrate-resistant acid phosphatases type-5 from the hairy cell leukemia spleen

Tartrate-resistant acid phosphatases types 5a and 5b were purified from human hairy cell leukemia spleen by sequential chromatography on Phenyl-Sepharose, CM-Sepharose, concanavalin A-Sepharose, FPLC Superose-12 and FPLC Mono-S. The purification over the original tissue extract was 1150- and 3300-fold, with a yield of 2.1% and 2.5%, respectively. Gel filtration indicated an Mr of about 30000 for both forms. There was a N-terminal sequence identity between the two enzymes. However, they appeared to be different as assessed by cation exchange chromatography and amino acid composition.     

Immunologic characterization of hairy cell leukemias in continuous culture.

The immunobiologic characteristics of three continuous cell lines established from hairy cell leukemia cells were investigated. All three cell lines continued to produce tartrate-resistant acid phosphatase, the enzymatic marker of hairy cells. Two of these cell lines were B lymphoid in nature. They carried Fc and C receptors, had surface and internal immunoglobulin, and did not form spontaneous sheep red blood cell rosettes. Experiments employing biosynthetic radiolabeling of immunoglobulin demonstrated distinctive immunoglobulin kinetics for each of these two hairy cell lines. One cell line remained quite similar to the original hairy cells from which it was derived whereas the other B lymphoid hairy cell line had undergone a switch in the immunoglobulin isotype produced. The third hairy cell leukemia line was shown to be of thymic derivation. These cells formed spontaneous sheep red blood cell rosettes and did not carry Fc or C receptors. The spontaneous sheep red blood cell rosette-forming cells contained tartrate-resistant acid phosphatase. They did not possess surface on internal immunoglobulin and did not synthesize immunoglobulin in vitro. Hairy cell leukemia cells maintained in permanent cell culture retain their immunobiologic properties and offer the opportunity for indepth study of these unusual cells.     

Flow cytometric analysis of hairy cell leukemia using right-angle light scatter

We report on an easy and reliable method for the enumeration of the typical cells present in the blood, bone marrow, and spleen of patients with hairy cell leukemia. Samples of five patients were analyzed. In three patients, the hairy cells were very accurately followed during treatment with alpha-interferon, using right-angle light scatter to differentiate them from other leukocytes present in total blood. Excellent correlation was obtained with the microscopic hairy cell enumeration. Additional verification by cell sorting and immunofluorescence studies confirmed these findings.     

Phorbol ester induction of plasmacytoid and hairy cell leukemia features in B-type lymphocytic leukemias: the relation to B-cell differentiation and maturation

Mononuclear cells concentrated from 11 patients with chronic lymphocytic leukemia (CLL), 7 with non-Hodgkin's lymphoma in leukemic phase (NHL), 5 with hairy cell leukemia (HCL), 1 with prolymphocytic leukemia (PLL), and 1 with plasma cell leukemia (PCL) were induced to differentiate with various doses of TPA. The degree of induction was followed for up to 6 days by measuring the expression of surface membrane markers (SmIg and GP-70) and Ig secretion, the induction of tartrate-resistant acid phosphatase (TRAP) and by recording ultrastructural changes as seen by electronmicroscopy. The results show a dose and time dependency of the TPA effect and a great heterogeneity in the cellular response, particularly in cells obtained from B-CLL patients. TPA induced two main features, namely the development of "plasmacytoid" or "hairy cell" leukemia features that clearly depended on the dose and duration of treatment with the phorbol ester. The plasmacytoid features were more frequently encountered with lower doses (1 ng/ml) of TPA and were more evident after shorter exposures to TPA (1-2 days). Nevertheless, the hairy cell features were more striking after incubation with higher concentrations of TPA (10-100 ng/ml) after longer periods of incubation (up to 6 days) with lower doses of TPA. The various features of differentiation measured including cell morphology, surface membrane markers, Ig secretion, and TRAP staining, were frequently independent of each other, suggesting an autonomous pathway of differentiation for some of these features. Furthermore, in most of the cases, hairy cell leukemia features were obtained more frequently following TPA exposure than plasmacytic changes.     

Purification and N-terminal amino acid sequence of the tartrate-resistant acid phosphatase from human osteoclastoma: evidence for a single structure

Tartrate-resistant acid phosphatase type-5 was purified to apparent homogeneity from human osteoclastomas by sequential chromatography on CM-Sepharose, Phenyl-Sepharose, concanavalin A-Sepharose, FPLC Superose-12, and FPLC Mono-S. The purification over the original tissue extract was 1167-fold, with a yield of 16%. An identity in the N-terminal amino acid sequence and Mr was found between this enzyme and two type-5 tartrate-resistant acid phosphatases isolated from hairy cell leukemia spleen. However, they appeared to be different as assessed by amino acid composition. In contrast to a previous report, no evidence was found for two subunits of the tartrate-resistant acid phosphatase.     

Electrophoretic study of lymphocytes from a patient with hairy cell leukemia

The electrophoretic mobility of lymphocytes is identified in the peripheral blood and from the spleen taken from a patient with hairy cell leukemia. Lymphocytes reveal an unimodal distribution with a low mobility in the histogram. There are no differences between the results obtained by investigating the peripheral blood and those of the spleen. Hairy cells could be delimited from lymphocytes of chronic lymphatic leukemia and immunocytoma by determining EPM.     

In vivo and in vitro induction of (2'-5') oligoadenylate synthetase by human interferons in leukocytes from healthy donors and patients with renal cancer and hairy cell leukemia

The effect of in vivo administered interferon-alpha (IFN-alpha) on 2-5-oligoadenylate (A) synthetase activity of peripheral blood mononuclear cells (PBMC) was compared in patients with hairy cell leukemia and renal cell cancer. Basic levels of this enzyme varied from donor to donor, but mean levels were not significantly different in patients with renal cell cancer or hairy cell leukemia compared to healthy donors. After a single injection of 3 x 10(6) IU IFN, these basic levels rose 2- to 8-fold within 12-24 h post-injection and reverted to pretreatment levels after 48 h. The extent of this in vivo stimulation by IFN-alpha was similar in patients with hairy cell leukemia and renal cell carcinoma, and was correlated with down-regulation of IFN-alpha receptors. The in vitro effects of IFN-alpha, -beta and -gamma were compared after 18 h treatment with 10, 10(2) and 10(3) IU/ml of each IFN. Unlike IFN-alpha and -beta, IFN-gamma did not induce 2-5 A synthetase activity in either normal PBMC or hairy cells; these results were related to the effects of the three IFN on proliferative response of normal PBMC to phytohemagglutinin. Our data support the idea that 2-5 A synthetase activity is a marker of biological response to interferon treatment in human cancers.     

Clinical significance of tartrate-sensitive and tartrate-resistant acid phosphatase indicated from the study of their biosynthetic mechanism

The tartrate-sensitive prostatic acid phosphatase, bands 2 and 4, are found in the soluble cytosol, and absent in the polysome of the prostate, while the tartrate-resistant acid phosphatase band 5 is present in the polysome and the soluble cytosol of hairy cells. The mRNA isolated from the prostate catalyzes the incorporation of 3T leucine into a protein different from that of bands 2 and 4. On the other hand, the mRNA isolated from the hairy cells catalyzes the incorporation of 3T leucine into band 5. The different biosynthetic mechanism of these two types of acid phosphatases are discussed in light of their different clinical significance.     

Phenotypic modulation of chronic lymphocytic, prolymphocytic, and lymphoplasmacytic leukemia cells by TPA: induction of TRAP isoenzyme 5 and HD6 (CD22) antigen and enhancement of IgM messenger RNA

B-cell neoplasias such as CLL can be viewed as models of monoclonal populations restricted within discrete ranges of B-cell maturation. It is unknown whether other B-cell leukemias such as prolymphocytic leukemia (PLL), lymphoplasmacytoid immunocytoma (IC), and hairy cell leukemia (HCL) involve different B lineages or are malignant variants of B cells in successive stages of development along the same lineage. Therefore in vitro maturation was induced with the phorbol ester TPA in leukemic cell samples from 10 CLL, 4 PLL, and 4 IC patients. Morphologically, both plasmacytic and hairy cell-like phenomena were induced. The latter unexpected finding was confirmed by reaction with HD6 (CD22) antibody which stains HCL but is unreactive with plasma cells, multiple myeloma, and CLL cells. Tartrate-resistant acid phosphatase was demonstrated in TPA-cultured CLL, PLL, and IC cells, and the same isoenzyme band as in HCL was revealed by isoelectrofocusing. On the other hand, an increase of IgM messenger RNA was detected in up to 20% of the cells in CLL cultures by single-cell in situ hybridization with fluoro-chrome-labeled DNA probes. An abundance of IgM messenger RNA characterizes lymphoplasmacytoid cells as found in IC. Our data demonstrate that CLL, PLL, and IC can be induced to realize a common genetic program which bears characteristics of HCL indicating that these four entities are more closely related than previously thought.     

In vitro response of cells from three patients with hairy cell leukemia to a recombinant leukocyte interferon

Mononuclear cells from the peripheral blood of two patients and from the spleen of one patient, all of whom had hairy cell leukemia, were cultured with a recombinant human leukocyte interferon (RD alpha 2-IFN). The IFN was added at concentrations of 10, 100, 1,000, and 10,000 IU/ml, and the cells were cultured for 1, 3, and 7 days. A cytocidal effect of IFN was observed only on cultured cells from the spleen at day 7. Electron-microscopic observations demonstrated that RD alpha 2-IFN induced the formation of tubuloreticular structures (TRSs) and annulate lamellae (ALs) in hairy cells, as well as in co-isolated non-leukemic cells, from all three patients. Ultrastructural examination revealed a close proximity between ALs and TRSs in co-isolated non-leukemic cells. A variability with respect to the induction of TRSs in hairy cells was observed among the three patients. In two of the three patients, the percentage of hairy cells with TRSs increased with the duration of incubation and with the dose of IFN. In the third patient, few hairy cells showed TRSs after 7 days of incubation with IFN. Our findings indicate that leukemic hairy cells may be heterogenous in their response to IFN.     

Acid alpha-naphthyl acetate esterase in hairy cell leukemia cells and other cells of the hematopoietic system.

The activity of alpha-naphthyl acetate esterase at an acid pH (ANAE) was investigated in 10 cases of hairy cell leukemia. All 10 cases, including two cases with only a few tartrate-resistant acid phosphatase-reactive cells, revealed a moderate or strong ANAE reaction. There was a characteristic pattern of activity consisting of small, medium-size, or large distinct granules often distributed in a semicircle in the cytoplasm, but sparing the nucleus of hairy cells. This reaction pattern was not found in T cells, T cell-derived leukemias, normal B cells, a number of B-cell lymphomas, myeloid cells, myeloid leukemias (including monocyte-derived leukemias), reticulum cell sarcoma, or malignant reticulosis. The cells from some B-cell lymphomas and plasmacytoma showed a relatively homogeneous pattern of fine or moderately coarse ANAE-positive granules that was similar to that of hairy cells only in some cases of plasmacytoma. Thus, fine to coarse granular ANAE reactivity is characteristic of hairy cells and is of potential diagnostic value for hairy cell leukemia.     

Detection of tartrate-resistant isoenzyme 5 of acid phosphatase in the white blood cells by disc electrophoresis in a case of hairy cell leukemia

In a case of hairy cell leukemia the evidence of tartrate-resistant isoenzyme 5 of phosphatase (TRISP 5) developed to findings important for the diagnosis. The evidence was achieved by means of disc electrophoresis by separating the cell lysate of white blood cells. The percentage of TRISP 5 in the total activity of acid phosphatase in leukocytes amounted to 17.6%. In cytochemical respect the evidence of TRISP 5 proved to be negative. The classification of these findings and their differential-diagnostic significance are discussed.     

The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.     

Recombinant interferon alpha-2 modulates hairy cell phenotype "in vitro"

The "in vitro" effect of IFN-alpha on the phenotypic profile of atypical cells from 5 hairy cell leukemia patients was investigated in a 72 hr culture assay. Cytochemical investigations revealed a dramatic decrease in the cytoplasmic content of acid phosphatase and tartrate resistant acid phosphatase in the absence of any apparent morphological modification. Flow cytometry showed that IFN-alpha markedly reduced the density of surface Ig without modifying the original isotype pattern. The expression of the receptor for the Fc fragment of IgG was also reduced. The class II MHC antigen recognized by the monoclonal antibody 12 remained essentially unchanged. Hairy cells were negative for OKT10 and PCA-1 and remained so after IFN-alpha incubation. Present data indicate that IFN-alpha is able to consistently and selectively affect membrane and cytoplasmic features of hairy cells in a short term period. The possibility is envisaged that these changes may be related to the therapeutic efficacy of IFN-alpha.     

Molecular analysis of the human chromosome 5q13.3 region in patients with hairy cell leukemia and identification of tumor suppressor gene candidates.

The pathogenesis of hairy cell leukemia (HCL) remains largely unknown since no specific genetic lesion has been identified in this disease. Previous cytogenetic analysis from our group has shown that chromosome abnormalities involving the 5q13 band are common in HCL, occurring in approximately 1/3 of patients. The data suggest that the 5q13.3 band is likely to harbor a gene involved in the transformational events of this disease. We have recently found two cosmids flanking the 5q13.3 breakpoint in patients with HCL, and the distance between them is approximately 35 kb, as analyzed by fiber-FISH. The two cosmids have been located between the markers SGC34998 and WI-15505/WI-6897 by radiation hybrid mapping. Five of 11 patients with HCL had a hemizygous deletion of the two cosmids, indicating that the function of a tumor suppressor gene may be lost. With the aim of delineating the critical region of 5q13.3 loss in patients with HCL, we have constructed an integrated contig of YAC, BAC, PAC, P1, and cosmid clones that covers the region. Within this area, three expressed sequences were identified as candidates for the putative 5q13.3 tumor suppressor gene involved in the pathogenesis of HCL.     

Purification and characterization of a tartrate-resistant acid phosphatase from human osteoclastomas.

Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.