Gerhard Gaedicke

Human leukemic cells: Receptor binding and biological effects of insulin and insulin-like growth factors

Receptor binding and biological effects of insulin and insulin-like growth factors I and II (IGF I/II) were assessed in three human malignant cell lines: a Burkitt-type ALL-cell line, a ANLL-cell line and a Hodgkins disease-cell line. Insulin receptor binding could be demonstrated in Burkitt-type ALL cells and ANLL cells, whereas no insulin receptor binding was detectable in Hodgkin cells. IGF I and IGF II binding could be demonstrated in all leukemic cells. Insulin stimulated glycogen synthesis in the insulin receptor bearing cell lines. DNA synthesis was stimulated by insulin, IGF I and II. IGF I was more active in stimulating DNA synthesis than IGF II.

Gamma-globulin therapy in 6 newly diagnosed diabetic children.

An autoimmune process may be involved in the pathogenesis of type I diabetes mellitus (1). Consequently, numerous therapeutic attempts for an early immunointervention have been carried out (2). Based on the favourable results with the gamma-globulin therapy in idiopathic thrombocytopenia (3), 6 newly diagnosed diabetic type I patients were treated with high doses of gamma-globulin to possibly prolong partial remission as defined by bikerblom (4). The pertinent clinical data at diagnosis and the results at follow-up are shown in Table 1. All patients were treated with human insulin (Lilly, FRG or Novo, Denmark). Within the first week of therapy the children received 0.4 glkg body weight Sandoglobulin@ (kindly provided by Sandoz, Nurnberg, FRG) on 5 consecutive days and the same dose once per week for up to 6 months. After discharge self-control was obtained with 3 daily urinary glucose tests and frequent blood glucose determinations (Diabur 5 OOO, Haemoglucotest 20-800, Boehringer, Mannheim, FRG). At intervals of 2-3 months C-peptide release was determined after an i.v.-glucose-arginene test or after a standard breakfast.

T-leukemia cell lines CCRF-CEM, HPB-ALL, JM and MOLT-4: Changes in isoenzyme profiles during induction of differentiation

SummaryBiochemical analysis has been used to monitor the induction of differentiation in cultured human T-leukemia cell lines (CCRF-CEM, HPB-ALL, JM and MOLT-4) by the phorbolester 12-O-tetradecanoylphorbol 13-acetate (TPA). The isoenzymes of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase were separated by isoelectric focusing on horizontal thin-layer polyacrylamide gels and stained by histo-cytochemical methods. TPA inhibited the proliferative activity in all four cell lines and led to aggregation of cells seen as floating clusters. TPA induced an increase in number and staining intensity of isoenzymes of all four enzymes in the cell lines studied. This corresponds to an induced isoenzymatic maturation as the progressive increase in number and staining intensity of the isoenzymes parallels the differentiation along the T-cell pathway. However, regardless of the initial stage of arrested differentiation, the cell lines could be induced only to differentiate to a certain more mature stage, but could not be triggered to differentiate terminally with regard to expression of isoenzyme patterns.

Occurrence of particular isoenzymes in fresh and cultured leukemia‐lymphoma cells. III. Esterase isoenzyme in monocytes

The expression of a particular α‐naphthyl acetate esterase isoenzyme which is specific for monocytes was examined in a panel of cultured leukemia‐lymphoma cell lines (n = 88), freshly obtained leukemia‐lymphoma cells (n = 527), and in fresh (n = 10) and cultured (n = 22) leukemia cells treated with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). The sodium fluoride‐sensitive isoenzyme was separated by isoelectric focusing on horizontal thin‐layer polyacrylamide gels. The esterase isoenzyme was not detected in untreated or TPA‐treated lymphoid, erythroid, or Hodgkins disease‐derived cell lines, but was seen in leukemia cell lines of monocytic origin. TPA induced the new expression of this marker isoenzyme in two leukemia cell lines of promyelocytic and erythroid origin that are known to differentiate along the monocytic‐macrophage cell lineage; TPA stimulation increased the staining intensity of the band in monocytoid cell lines. This esterase isoenzyme was found in 92% of the cases classified morphologically as acute myelomonocytic or monocytic leukemia, but only in 3% of the non‐monocytic acute myeloid leukemias. All lymphoid or erythroid leukemias or lymphomas were negative. Treatment with TPA of AML and CML cells, which commonly differentiate to monocyte/macrophage‐like cells, showed de novo the monocyte‐specific isoenzyme. It is concluded that this isoenzyme is a characteristic marker for monocytic leukemia cells and will be a useful tool for the discriminatory identification of the monocytic element in normal and leukemic cells. Cancer 59:77–82, 1987.

Expression of a monocyte-specific esterase isoenzyme in cases of acute myeloid leukemias.

The carboxylic esterase (E.C. 3.1.1.1) isoenzymes from cases of acute myeloid leukemias were separated by analytical isoelectric focusing on horizontal thin-layer gels. One isoenzyme consisting of one or two components (bands) could be completely and selectively inhibited by addition of 40 mM sodium fluoride (NaF) to the staining bath. The 105 cases were classified into the groups M1-M6 according to the FAB proposals. The NaF-sensitive isoenzyme was not detected in cases of FAB groups M1/2 (acute myeloblastic leukemia without or with maturation), group M3 (acute promyelocytic leukemia) or group M6 (erythroleukemia). Thirty-one out of 33 cases in the FAB group M4 (acute myelomonocytic leukemia) and 9/9 cases in FAB group M5 (acute monocytic leukemia) expressed the NaF-sensitive isoenzyme. The NaF-sensitive isoenzyme was found at different staining intensities; all M5 cases showed the isoenzyme at strong or very strong intensity, whereas most of the M4 cases displayed the isoenzyme at weak, medium or strong staining intensity. The data presented are further evidence that the presence of the NaF-sensitive esterase isoenzyme indicates monocytic involvement or differentiation in cases of myeloid leukemias. The easy and fast to perform method of isoelectric focusing can be used to distinguish the monocytic variants among the acute myeloid leukemias and can supplement the morphological analysis of these cases.

Conversion of acute undifferentiated leukemia phenotypes: analysis of clonal development.

The cellular origin of acute undifferentiated leukemia (AUL) is still a matter of controversy. We report on two cases in which the diagnosis of AUL was established according to restricted criteria. Blast cells of both patients showed phenotypic conversion during the course of disease. In one case, within 24 days from starting treatment, the leukemic phenotype changed from AUL to acute myelomonocytic leukemia (FAB L1, TdT+ to FAB M4, TdT-). The initial phenotype of this acute leukemia was characterized by the co-expression of both B-lymphoid and myeloid markers on the same cell. Moreover, analysis of esterase isoenzyme pattern showed the whole spectrum of isoenzymes typically seen in myelomonocytic leukemias already at diagnosis, yet blast cells additionally contained all three isoenzymes of beta-hexosaminidase typically seen in AUL. However, examination of immunoglobulin (Ig) heavy chain gene rearrangement initially and after conversion revealed an identical monoclonal configuration of Ig heavy chain sequences in both samples. The second AUL patient relapsed after allogeneic bone marrow transplantation with common ALL-antigen (CALLA) positive acute leukemia. Subsequent Southern blot analysis showed a novel rearranged Ig fragment compared to the analysis before transplantation indicating that the leukemic clones prior to and after transplantation were not identical. No chromosomal abnormalities were observed in both cases. These data support the view that AUL cells originate from a pluripotent stem cell that is capable to differentiate in the myelomonocytic lineage (patient 1), and confirm the value of Ig gene analysis as marker for cellular clonality.

Expression of FMC7 antigen and tartrate-resistant acid phosphatase isoenzyme in cases of B-lymphoproliferative diseases.

A panel of different B-cell malignancies representing various stages of B-cell differentiation were analyzed for the expression of an antigen labeled by the monoclonal antibody FMC7 and of tartrate-resistant acid phosphatase (TracP) activity. The FMC7 antigen and TracP were not found on early immature pre B-cell proliferations, appeared at early and intermediate B-cell stages, reached their peak of expression in terms of both incidence of positivity and staining intensity at the late B cell stage (as represented by hairy cell leukemia) and were lost at the B-cell/plasma cell transition. Although detected at similar stages of B-cell differentiation, FMC7 and TracP appear to be independently expressed and were not related to a particular Ig class. The simultaneous detection of FMC7 and TracP represents a distinguishing parameter for the identification of hairy cell leukemia.

ß-Hexosaminidase Isoenzyme I: An Early Marker of Hematopoietic Malignancy

The analysis of various enzymes has been found to distinguish immunologic subsets of human leukemias. This is especially true for terminal deoxynucleotidyl transferase (TdT). This enzyme is present in 90% of all cases with common ALL, pre B-ALL, T-ALL, and it is negative in B-ALL. Some 5%–15% of AML are also positive for TdT. Interestingly, acute undifferentiated leukemias (HLA-DR positive, cALL-A negative) have been found to be TdT positive in 40%–60% of cases. This nuclear enzyme thus seems to be a very early marker of lymphohematopoietic development. The lysosomal enzyme s-hexosaminidase (N-acetyl-s-D-glucosaminidase) can be separated into two major forms: an isoenzyme A, which is constituted by two a and two s subunits, and in isoenzyme B, which consists of four s subunits.

Reactivity patterns of monoclonal antibodies positive on myelomonocytic leukemia cells as defined by esterase isoenzyme analysis

SummaryThe reactivity with monoclonal antibodies (MoAbs) specific for myelomonocytic cells and the expression of a particular esterase isoenzyme were analyzed in 159 cases of acute myeloid leukemias. The incidence of positivity of 16 MoAbs (MCS-2, MCS-1, OKM1, My-1, Leu-M1, Leu-M3, CA-2-38, MY4, MY7, MY8, MY9, VIM-D2, VIM-D5, Mo1, Mo2, 63D3) was studied using the indirect immunofluorescence technique. A carboxylic esterase isoenzyme which can be inhibited completely and selectively by sodium fluoride (NaF) was demonstrated by isoelectric focusing on horizontal polyacrylamide gels. This NaF-sensitive isoenzyme indicated the monocytic origin of the blast cells as it is specific for this cell lineage. Prior to the immunological-isoenzymatic analysis all cases were categorized into two subtypes according to morphological criteria of the FAB classification system: 147 cases of AML (FAB M1-3) and 12 cases of AMMoL/AMoL (FAB M4/5). However, 15 out of 147 cases of AML expressed the NaF-sensitive isoenzyme and were therefore assigned to the group AMMoL/AMoL. Likewise, 1 case, diagnosed morphologically as AMMoL, was negative for this marker isoenzyme and was assigned to the other leukemia subtype. The incidence of reactivity varied widely for the MoAbs tested regarding the overall results on all cases and the positivity on cases of either AML or AMMoL/AMoL. The MoAbs were grouped into four classes depending on the pattern of reactivity with myeloblastic or monoblastic or both subtypes of acute myeloid leukemia. The MoAbs MCS-2, MY7, Leu-M1, and MY9 detected the vast majority of cases with either myelocytic or monocytic involvement (group-I: “pan-myelomonocytic” reactivity). The MoAbs MCS-1, OKM1, VIM-D5, and Mo1 showed a predominance in their staining pattern for monocytic variants, but were also positive on a substantial percentage of nonmonocytic cases (group-II: predominantly reactive with monocytic, but also myelocytic cases). The MoAbs Leu-M3, MY4, VIM-D2, Mo2, and MY8 reacted with the large majority of AMMoL/AMoL cases and with a small number of AML cases (group-III: monocyte-“specific” reactivity). The MoAbs of group-I are useful in differentiating acute lymphoid from acute myeloid leukemias. The MoAbs of group-III, and to a lower extent those of group-II, will be of considerable value in the subtyping of acute myeloid leukemias. The results show that (1) accuracy of leukemia classification might not always be achieved by morphology alone, but that immunological and biochemical aspects should be included as well, and (2) several MoAbs are very useful tools for classification and subtyping of acute myeloid leukemias.

Changes in Isoenzyme Patterns Expressed by the Erythroleukemia Cell Lines K-562 and HEL After Induction of Differentiation

Stable human leukemia-lymphoma cell lines provide model systems to study the processes involved in leukemic and normal cell differentiation [10]. Leukemic cells, arrested at a certain stage of differentiation, can be triggered to differentiate to functionally and morphologically more mature cells [7]. Both “fresh” leukemic cells and cells maintained in long-term culture are sensitive to in vitro induction of differentiation. Furthermore, cell differentiation is a novel concept in the treatment of acute leukemias and several substances such as low dose Ara-C and the physiologic Compound retinoic acid have been shown to act as differentiating agents in vivo [7].