Acetylcholinesterase in cultured human leukemia/lymphoma cell lines

Fifty-two cultured leukemia/lymphoma cell lines were studied for their acetylcholinesterase activity. There was a striking effect of maturity on enzyme activity, only the most mature cells showing significant activity. Mature T cells exhibited far more enzyme activity than mature B cells, paralleling results on normal T and B cells.     

Characterization of thymocytes expressing the common acute lymphoblastic leukemia antigen

We studied the relationship between CALLA + thymocytes and two known markers of T-lymphocyte differentiation, Tdt and the sheep erythrocyte receptor. Thymocytes were studied using double fluorochrome analysis (with monoclonal anti-CALLA antibody and anti-Tdt) before and after E rosette separation. We found that approx. 4% of unseparated thymocytes were CALLA + and that most CALLA + cells were also Tdt+. After E rosettes separation CALLA + Tdt + cells were found mostly in the ER- fraction (20% of ER- cells) while only 1.0% of ER + cells were CALLA +. The expression of CALLA on ER? Tdt + thymocytes suggests that CALLA may defined cells early in T-cell differentiation.     

A new set of monoclonal antibodies against acute lymphoblastic leukemia

Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.     

Immunodiagnosis of childhood ALL with monoclonal antibodies to myeloid and lymphoid associated antigens

Sixty-five cryopreserved leukemic samples from children diagnosed and treated as having acute lymphocytic leukemia (ALL) were retrospectively examined for the presence of lymphoid and myeloid associated antigens by indirect immunofluorescence using monoclonal antibodies. Expectedly, the majority of these specimens expressed antigens known to be expressed on lymphoid, and not myeloid malignancies. These included the common acute lymphoblastic leukemia antigen (CALLA), the p32 B-cell associated antigen, and T-cell associated antigens. Leukemic cells from the 8 remaining patients expressed antigens known to be present on both myeloid and lymphoid leukemias. These included HLA/DR, and the antigens identified by BA-1 and BA-2. Cells from 2 of these 8 patients reacted with antibodies that define antigens present on normal and malignant myeloid cells. Both specimens reacted with 1G10, an anti-granulocyte antibody, and one reacted with 5F1 which reacts with monocytes, nucleated red blood cells, megakaryocytes and platelets. One of these patients relapsed while receiving ALL therapy, and the morphology of her leukemic cells became characteristic of acute monocytic leukemia (AMoL). The second patient failed ALL therapy but responded to standard acute nonlymphocytic leukemia (ANLL) therapy, clearing her peripheral blasts. Thus these studies confirm that cell surface phenotyping with monoclonal antibodies can recognize ALL cells that express myeloid rather than lymphoid associated antigens and demonstrate that the malignant cells display a clinical behavior consistent with the diagnosis of ANLL.     

Isoenzyme studies in human leukemia-lymphoma cell lines--1. Carboxylic esterase

The isoenzyme patterns of carboxylic esterase (E.C. 3.1.1.1) were studied in 74 proven human leukemia-lymphoma and 12 normal B-lymphoblastoid cell lines. These cell lines have been extensively phenotyped using poly- and monoclonal antibodies. Esterase isoenzymes were separated by isoelectric focusing and visualized by histo-cytochemical techniques. No leukemia-specific or (except for monocytes) blood cell type-specific isoenzyme or isoenzyme pattern could be detected. The monocytic element in some cell lines was characterized by a strong isoenzyme band which could be selectively and completely inhibited by sodium fluoride. The enzyme phenotypes were stably expressed in all subcultures of a given cell line and did not appear to have any cell cycle dependency. The leukemia-lymphoma cell lines have been subclassified into four major groups according to immunological parameters: T-cell, B-cell, myelomonocytic and non-T, non-B-cell. On the basis of immunological data the T-cell lines were assigned to five stages of differentiation. The number and staining intensity of the isoenzymes increased with differentiation of the T-cells paralleling the expression of immunological markers. The B-cell leukemia-lymphoma cell lines were divided into pre B-, B-, Burkitt lymphoma, multiple myeloma and hairy cell leukemia cell lines. Substantial variability among the isoenzyme patterns was detected ranging from immature profiles of pre B-cell lines to complete isoenzyme repertoires of multiple myeloma cell lines. No significant difference was seen between the isoenzymes of mature B-cell lines and normal B-lymphoblastoid cell lines. The most prominent feature seen in myelomonocytic cell lines was the monocytic band indicating a monocytic origin and separating the 'monocytoid' from the 'pure myeloid' cell lines. Considerable heterogeneity in the isoenzyme patterns was observed in the non-T, non-B cell groups which comprised erythroleukemia cell lines and cell lines arrested at a very early stage of lymphoid differentiation. These latter cell lines together with some T- and B-cell lines shared the common characteristics of positivity for cALLA, TdT and Ia antigens and an immature, incomplete isoenzyme profile. The results support the notions of maturation arrest and normal gene expression in leukemic cell populations. Furthermore, the importance of biochemical studies as part of the multiple marker analysis could be demonstrated.     

B-lymphocyte associated differentiation antigen expression by 'non-B, non-T' acute lymphoblastic leukemia

We investigated the neoplastic cells obtained from 37 cases of 'non-B, non-T' (SIg-E-) acute lymphoblastic leukemia (ALL) for their expression of 13 distinct monoclonal antibody defined B lymphocyte associated differentiation antigens. We correlated the expression of these B cell antigens with terminal deoxynucleotidyl transferase (TdT), HLA-DR antigen, common ALL antigen (cALLa), and cytoplasmic mu heavy chain (Cu) expression by these neoplastic cells. In this way, we were able to describe a hierarchy of B lymphocyte associated differentiation antigens as well as the marked phenotypic heterogeneity of 'non-B, non-T' ALL. TdT and HLA-DR are expressed throughout the stages of B cell differentiation represented by 'non-B, non-T' ALL. The earliest B cell antigen appears to be Leu 12 (B4) followed by BA-2 and then BL2. OKB2, BL1 and BA-1 are acquired next, followed by B1, BL3, cALLa and Cu. BL7 appears just prior to SIg. OKB1, OKB4, OKB7 and BL4 appear at or after the time of SIg expression and hence are not expressed by 'non-B, non-T' ALL cells. This developmental hierarchy is supported by the results of phorbol ester (TPA) induction studies. Thus, cases of 'non-B, non-T' ALL constitute a useful model for probing the hierarchal expression of B cell antigens and delineating the B cell developmental pathway(s).     

Cytochemical distribution of dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.5) in T-lymphoblastic lymphoma/leukemia characterized with monoclonal antibodies

In human blood and bone marrow, dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.5) selectively occurs in T lymphocytes bearing Fc receptors for IgM. In the present study 35 cases of lymphoblastic lymphoma and leukemia were analysed for the specificity, incidence and reaction pattern of DAP IV. On the basis of immunohistochemical staining with monoclonal antibodies and enzyme cytochemical staining for acid phosphatase, 12 cases were classified as B-type neoplasms. In 23 cases T-cell properties were expressed to different extents, apparently reflecting different categories of maturation. Whereas B-cell lymphomas were invariably negative for DAP IV, seven of the 23 T-lymphoblastic lymphomas/leukemias showed this enzyme. Thus DAP IV is a highly specific marker for a distinct T-cell subpopulation, apparently irrespective of the stage of differentiation.     

Distinct lymphoblastic and myeloblastic populations in TdT positive acute myeloblastic leukemia: evidence by double-fluorescence staining

Double-immunofluorescent staining for the enzyme terminal deoxynucleotidyl transferase (TdT) as a marker of primitive lymphoblasts, and for the VIM-D5 antigen as a differentiation antigen of the myeloid system gave direct evidence for distinct lymphoblastic and myeloblastic populations (mixed leukemic cell populations) in seven patients with acute leukemia. The percentage of malignant TdT positive cells contributing to a leukemic cell bulk with unequivocal signs of myeloid origin was between 10 and 80%. A defect at the level of a common progenitor cell giving rise to both the TdT and the VIM-D5 positive blast cell population is discussed.     

Differentiation-associated changes in human non-T, non-B leukemia cell lines after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA)

The ability of TPA to induce stable phenotypic changes that normally serve as markers of differentiation was examined in the four human non-T, non-B cell lines, NALL-1, NALM-16, REH and KM-3. In all four lines, noncytotoxic concentrations of the phorbol ester caused an extensive reduction in the number of cells expressing cALL surface antigen and terminal deoxynucleotidyl transferase. The disappearance of these markers correlated with the loss of cell proliferation. In one of the cell lines, NALL-1, TPA treatment gave rise to a significant increase in Ia-like antigen and antigen T-101, markers which represent more advanced stages of cell maturation. However, surface or cytoplasmic immunoglobins, indicators of mature B cells, were not detectable. Antigen 3A1, specific for myeloid and for T cells, antigen Leu-4, specific for T cells and antigen CM1, specific for monocytes, were also absent. In all cell lines, exposure to TPA resulted in an approximately two-fold increase in acid phosphatase and beta-glucuronidase activity. The emergence of these phenotype changes was not altered upon repeated washing of the TPA-treated cells. These results demonstrate that while TPA is capable of inducing various non-T, non-B cell lines to differentiate to a limited degree, differences exist between the lines in the extent to which they can mature towards the B-cell stage.     

A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells

Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.     

Isoenzyme studies in human leukemia-lymphoma cell lines--IV. Lactate dehydrogenase

Isoelectric focusing (IEF) in horizontal polyacrylamide gels has been used to separate lactate dehydrogenase (LDH) isoenzymes in 97 human permanent hematopoietic cell lines (85 leukemia-lymphoma cell lines and 12 'normal' B-lymphoblastoid cell lines). Maximally 8 LDH bands were seen; the electrophoretically detectable bands 4 and 5 could be separated by IEF into 2 and 3 isoenzymes, respectively. The LDH patterns have been found to vary both in number of isoenzymes and in relative intensity in different cell lines depending upon the stage at which arrest of differentiation occurred. These differences can be used to analyse and distinguish different cell lines. The method should provide a valuable supplement to the enzymatic phenotyping and complete characterization of fresh and cultured leukemias and for the monitoring of phenotypic changes occurring during induction of differentiation.     

Acute undifferentiated leukemia: induction of partial differentiation by phorbol ester

Expression of lineage-associated surface antigens, was studied in 7 patients with acute undifferentiated leukemia (AUL), 3 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoid leukemia (ALL) and bone marrow from 2 healthy donors, before and after exposure to the differentiating agent 12-O-tetradecanoylphorbol-13-acetate (TPA). The surface antigens were identified by monoclonal antibodies (My4, My8, My9, MO1, B1, CALLA, T11) and formation of EA and EAC rosettes. Adherence to plastic was also assessed. Cells from the AML patients responded to TPA with an increase in myeloid antigen positive cells and other markers of differentiation. Four of the AUL patients showed, also, a large increase in the fraction of cells expressing one or more myeloid markers, in correlation with formation of EAC rosettes. In contrast, the percentage of cells expressing myeloid antigens, did not increase in the 4 ALL patients, or in the normal donors. These findings confirm the heterogeneity of undifferentiated leukemias, and suggest the hypothesis that some AUL's can be induced to express markers of early myeloid cells.     

Plasmacytoid blast crisis in B-cell chronic lymphocytic leukemia: effect of estradiol on growth and differentiation in vitro

Evolution of a case of chronic lymphocytic leukemia (CLL) into blast crisis was found to be characterized by three unusual features (1) the phenotype of the emerging blast cells was that of pre-plasmacytoid cells as shown by plasma cell morphology and an immunological phenotype corresponding partially with CLL- or intermediate B-cells, partially with plasma cells (terminal transferase-, common acute lymphocytic leukemia antigen-, Ia+, surface immunoglobulin heavy chains-, surface kappa light chains+, intracytoplasmic immunoglobulin A+ and G+, BA-1+, polyclonal gammaglobulin production); (2) cytogenetic analysis of spontaneous metaphases revealed that in addition to the typical CLL abnormality, trisomy 12, in all of the cells, an additional translocation between chromosomes 14 and 17 was present in 40% with a presumptive breakpoint on chromosome 14 (q12-3) never described before (commonly q32) and (3) the progression of the disease was associated with a striking increase in the expression by the transformed cells of specific binding sites for estradiol (E2) due to an actual increase in total cellular receptor proteins and not to a change in receptor affinity for E2. The functional status of the steroid receptors was confirmed by nuclear transfer of the cytoplasmic hormone-receptor complex upon temperature activation. Since the rise in E2-receptor display paralleled a large increase in the proliferative activity of the cells as well as a change in their maturation status the question was raised as to whether the E2-receptor should be considered as a physiological marker of growth rate or of cellular differentiation. Exposure of the patient's blast cells to E2 in vitro resulted in cessation of cell growth following at least one mitosis after addition of the inducer as seen from the replacement of the large blasts by small CLL-like cells without definite signs of alteration of the differentiation status. This suggests the association of E2-receptor expression with control of growth rather than cell maturation.     

Isoenzyme studies in human leukemia-lymphoma cell lines--III. beta-Hexosaminidase (E.C. 3.2.1.30)

The hexosaminidase (beta-N-acetylglucosaminidase) isoenzyme profiles of 86 human hematopoietic cell lines grown actively in suspension culture were analysed by isoelectric focusing and by conventional disc electrophoresis on horizontal thin-layer polyacrylamide gels. A maximum of three hexosaminidase (Hex) isoenzymes (A = anodic, I = intermediate, B = basic) could be demonstrated. The immunological phenotyping of 74 leukemia-lymphoma derived cell lines had led to a categorization into four groups with a subclassification of the T- and B-cell lines into several stages of differentiation: 26 T-cell, 34 B-cell, 6 myelomonocytic and 8 Non-T, Non-B cell leukemia-lymphoma cell lines. Twelve so-called 'normal' B-lymphoblastoid cell lines were also available. Distinct isoenzyme profiles were seen in the different stages of differentiation in the T- and B-leukemia-lymphoma cell lines. Among the 12 normal B-lymphoblastoid cell lines heterogeneity in the isoenzymatic phenotypes was detected. Hex isoenzyme expression in normal and neoplastic lymphoid cell lines represents hypothetically sequential stages of T- and B-cell differentiation. Myelomonocytic cell lines displayed strongly stained bands of all three isoenzymes. Heterogeneity was seen in the group of Non-T, Non-B cell lines. Four out of 5 pre B-cell lines and 4 out of 4 Non-T, Non-B cell lines which are comparable to cases of pre B- and common ALL revealed a high Hex I/Hex A ratio in terms of intensity of the isoenzyme bands. The analysis of Hex isoenzymes is useful for characterizing lymphoid and myeloid populations (both normal and malignant, cultured or fresh), particularly with regard to their stage of differentiation. But this enzyme should be part of a multiple enzyme study where the information obtained is complementary. In turn, enzyme marker analysis should be included in the multiple marker analysis for an optimized characterization of leukemic cells.     

Terminal transferase immunofluorescence, enzyme markers and immunological profile of human leukemia-lymphoma cell lines representing different levels of differentiation

We have examined alterations in terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) in MOLT-4 cells during changes in growth conditions. Subsequently, we used cells from 48 human hematopoietic cell lines of different cell lineages and maturation stages to compare the IF and biochemical assays for expression of TdT. In addition, we have attempted to correlate the expression of TdT, adenosine deaminase (ADA), thymidine phosphorylase (TP) and immunological markers with maturation stages in these different cell lines. The results indicate that TdT positive cells remain TdT positive when assayed by either the biochemical or IF tests during growth or early plateau phase, but that cells under poor growth conditions, such as in old cultures, may give a negative TdT IF reaction. Otherwise, biochemical and IF assays for TdT gave comparable results in the 48 cell lines tested, testifying to the reliability of the IF test. Based on the comparisons of the various cell lines studied, it appears that both ADA and TdT decrease progressively as maturation of T cells from Blast I to Blast IV to mature T cells increases. TP was deficient in all T-cell lines compared to normal peripheral blood T cells, which in turn had lower activity compared to normal peripheral blood B cells. Pre-B cells, although indistinguishable from each other by immunological markers and all having low TP and ADA activity, showed heterogeneity, with TdT activity high in some and low in others. All non-T, non-B lines had high TdT activity, but low ADA and TP activity. B- and myelocytic cell lines had low ADA and TdT activity, and showed an increase in TP activity as the maturation of cells increased. These results indicate that the TdT IF test is a reliable procedure for detecting TdT positive cells, and that TdT, ADA and TP could be useful markers for studying the differentiation of human hematopoietic cells.     

Patient with acute B-cell leukemia of burkitt's type (L3) and marker chromosomes including an (8;14) translocation

A 20-year-old man with acute B-cell leukemia of Burkitt's type (L3) presenting unusual début symptoms with jaw involvement is reported. The leukemic cells revealed chromosomal abnormalities including four marker chromosomes [1q+, 6q?, t(8;14)].     

The demonstration of terminal deoxynucleotidyl transferase on frozen tissue sections and smears by the avidin-biotin complex (ABC) method

A method using avidin-biotin complex (ABC) to detect the presence of the enzyme terminal deoxynucleotidyl transferase (TdT) is described and compared with a proven indirect immunofluorescence method. The material studied consisted of: (1) peripheral blood and bone marrow smears from 17 patients with leukemia (ALL, 8; CLL, 3; AML, 6), six normal controls, one T-ALL cell line, and (2) frozen tissue sections from four patients with lymphoblastic lymphoma (LL), two patients with nodular poorly differentiated lymphocytic lymphoma (NLPD), two patients with reactive follicular hyperplasia (RFH) and one calf thymus. The slides from patients with ALL had from 30 to 90% cells with nuclear positively by the ABC technique. Slides from patients with CLL were negative, as were the normal peripheral blood smears. Normal bone marrow smears contained less than 5% positive cells. The T-ALL cell line was 100% positive. The frozen tissue sections from the patients with LL and the calf thymus contained numerous positive cells, while all of the sections from the patients with NLPD and RFH were negative. A good correlation existed between the ABC and the indirect immunofluorescence methods. The ABC method described is both more specific and more sensitive than the previously described techniques in detecting TdT in tissue sections and smear preparations.     

Isoenzyme studies in human leukemia-lymphoma cells lines--II. Acid phosphatase

This report describes the qualitative acid phosphatase (acP) isoenzyme profiles detected in permanent human hematopoietic cell lines. The acP activity was separated into its isoenzymes by isoelectric focusing on horizontal thin-layer polyacrylamide gels. The pattern of acP isoenzyme was investigated in a total of 86 cell lines. These cell lines were classified into five groups on the basis of their phenotypes characterized in the multiple marker analysis: 74 leukemia-lymphoma cell lines (26 T-, 34 B-, 6 myelomonocytic, 8 Non-T, Non-B cell lines) and 12 so-called 'normal' Epstein-Barr virus transformed B-lymphoblastoid cell lines. Their immunological features had been analysed in detail by use of a large panel of poly- and monoclonal antibodies which led to a further subclassification into stages of differentiation. A progressive increase in number and staining intensity of the isoenzymes which paralleled the expression of surface markers at different stages of differentiation along their developmental pathway was seen in the T- and B-leukemia-lymphoma cell lines. Some cell lines whose isoenzyme profiles did not correspond to the stage of differentiation as evidenced by surface antigen analysis might represent good examples of deranged gene expression in otherwise normally programmed malignant cells, i.e. in our study a mismatch between the isoenzymatic and immunological phenotypes. The tartrate-resistant isoenzyme was detected in 9 out of 74 leukemia-lymphoma cell lines (4 T-, 2 B-, 1 myelomonocytic, 2 Non-T, Non-B cell lines) and in 10 out of 12 normal B-lymphoblastoid cell lines; the only one studied hairy cell leukemia cell line did not express this isoenzyme. The relative specificity of the tartrate-resistant acP is discussed in detail. No leukemia-lymphoma specific isoenzyme or an additional isoenzyme which was not seen in normal hematopoietic cells could be observed. Nor did we find an isoenzyme or isoenzyme pattern characteristic for a certain cell lineage. This underlines the necessity of a combined analysis using markers from different disciplines in the 'multiple marker analysis' in order to accurately characterize normal and malignant blood cells. Furthermore, our results support the concept of maturation arrest at particular stages of differentiation together with the theory of normal gene expression in leukemic cells equivalent to that in their normal counterparts.     

Surface antigen expression by a human B-lymphoblastoid cell line treated with 'differentiation' inducers, dimethysulfoxide and tetradecanoylphorbol acetate

Changes in expression of surface antigens and morphology induced in a human B lymphoblastoid cell line by 'differentiation' inducers DMSO and TPA have been examined. Both of these agents were shown to cause arrest in the G1 phase of the cell cycle, and morphological changes including a decrease in the nuclear to cytoplasmic ratio. In addition, TPA caused a marked increase in membrane area with extensive ruffling. Treatment of cells with 1.25% DMSO for 6 d brought about decreases in the expression of Ia antigens, CALLA, and an immature cell antigen defined by the monoclonal antibody 11D1, and an increase in the expression of surface membrane immunoglobulin. This pattern is consistent with the induction of differentiation towards a more mature B cell. In contrast, treatment of cells with 5 X 10(-8)M TPA for 2 d resulted in increased expression of both Ia and HLA-A, B,C antigens, decreased expression of surface membrane immunoglobulin, and little or no change in the other markers. These changes do not indicate a maturation process and can be explained in part by accumulation of cells in the G1 phase of the cell cycle.