Purine metabolism in childhood acute lymphoblastic leukemia: biochemical markers for diagnosis and chemotherapy

Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), 5'nucleotidase (5'NT), ecto-5'NT, hypoxanthine-guanine phosphoribosyltransferase(HGPRT), adenine phosphoribosyltransferase(APRT), adenosine kinase(AK), AMP deaminase (AMPD) and adenylate kinase(AdKin) activities were assayed in leukemic cells from bone marrow and/or peripheral blood of 43 newly diagnosed children with acute lymphoblastic leukemia(ALL). These enzyme activities have been investigated in relation to some immunological markers. ADA activity was higher in E-rosette positive leukemia(E+ ALL), while HGPRT, APRT, PNP, 5'NT, ecto-5'NT and AdKin activities were found to be lower in E+ ALL as compared to E- ALL. In common ALL (cALL) antigen positive leukemia, mean ADA activity was significantly lower as compared to cALL- leukemia, whereas PNP, 5'NT, ecto-5'NT and AdKin activities were significantly higher. cALL cells with cytoplasmic immunoglobulin M(IgM) heavy chains were found to have mean 5'NT activities twice as high as cALL cells lacking cytoplasmic IgM heavy chains. In two patients who had surface immunoglobulins on their cell membranes, low 5'NT activities were found. When measuring enzyme activities after 2-4 days of prednisone monotherapy, only mean ADA and HGPRT activities decreased in non-B, non-T ALL. These decreases were not significant in T-ALL patients. Mean enzyme activities in the leukemic cells of five patients with relapse were comparable to those in newly diagnosed patients, except for 5'NT, which was found to be within the activity range of control peripheral blood lymphocytes. It is concluded that ADA and AdKin activities are suitable as markers for E+ ALL and cALL+ leukemias respectively. 5'NT might help to distinguish between cALL cells having and lacking pre-B characteristics. Since 5'NT activity may also be decreased in B-ALL, it is not suitable as a T-ALL marker. Enzymes of purine metabolism in leukemic relapse need further investigation.     

Prognostic value of 5'nucleotidase in acute lymphoblastic leukemia with the common-ALL phenotype

5'-Nucleotidase (5'NT) is an enzyme found on the surface membrane of leukemic cells with the c-ALL phenotype. We studied 79 children with acute lymphoblastic leukemia (ALL). Thirty six of them had the c-ALL phenotype. Within this c-ALL group, ten had 5'-NT positive leukemic cells. Clinical data were available in 33 c-ALL cases. Sex, age and initial white blood cell count were comparable between 5'NT positive and 5'NT negative c-ALL cases. In the 5'NT positive group the probability of complete continuous remission was significantly lower than in the 5'NT negative group (p less than 0.05).     

Detection of binding sites for spiroperidol on leukemic cells: its value for the phenotype characterization of lymphoid leukemias

The specific binding of the dopamine antagonist spiroperidol was studied in leukemic cell samples of various phenotypes. Among these only B-cell samples from chronic lymphocytic leukemias ($\text{7}\text{7}$) and some “null” cell samples from acute lymphoblastic leukemias ($\text{2}\text{7}$) showed specific binding. B cells from a prolymphocytic leukemia were negative as were also T-lymphoïd and non-lymphoïd leukemc cells at different stages of maturation. This pattern can be clearly correlated with the previous results obtained with normal blood cells and on cell lines. Moreover, it suggests that the detection of spiroperidol binding sites could provide a new means of distinguishing different phenotypes among B cells and early lymphoïd cells.Our results open the way to further studies which might show a correlation between spiroperidol binding sites and the new immunological markers defining subsets among non-T lymphoïd cells, as well as defining their physiological meaning.     

Deoxycytidylate deaminase activity in lymphoproliferative disorders

The activity of dCMPase has been measured in cell extracts from human lymphoproliferative disorders. The highest levels occurred in T helper-CLL and Thy-ALL, but high levels were also found in C-ALL, NPDLL transforming to DHL, DPDLL and DHL. A range of enzyme activities was found in the majority of types examined, with the widest range encountered in ALL, NPDLL and DHL. In DWDLL, a narrow range of dCMPase activities was found, with enzyme levels in the control range or moderately increased. Similarly, B-CLL exhibited a narrow range of enzyme activities, within that of the controls. The highest enzyme activity in HD was found in the highly malignant type — lymphocyte depleted HD. Statistically significant differences were found between the distribution of dCMPase activities in ALL and the chronic leukemias; and between favorable histologic types of non Hodgkin's lymphomas and the unfavorable DHL type. These data suggest that dCMPase activity is a marker of the clinical aggression of human lymphoid malignancies. Moreover, the marked variation in enzyme activity in each type of lymphoid malignancy suggests that this also applies to individual tumors as well. In view of the important role of dCMPase in pyrimidine metabolism and the profile of enzyme activities in leukemia and lymphoma, it is suggested that an inhibitor of dCMPase could be of clinical value in lymphoid malignancy.     

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.     

Clofarabine: past, present, and future

Clofarabine is a second generation purine nucleoside analogue designed to overcome the limitations and to incorporate the best qualities of both cladribine and fludarabine. Clofarabine is thought to work via three mechanisms: inhibition of ribonucleotide reductase; incorporation into DNA; and induction of apoptosis. Given these mechanisms of action, clofarabine would be predicted to act synergistically with other chemotherapeutic agents such as other purine nucleoside analogues and DNA damaging or cross linking agents such as anthracyclines and platinum-based compounds. Intravenous clofarabine showed significant efficacy in pediatric leukemias (specifically, acute lymphoblastic leukemia (ALL)) and, in 2004, it was approved by the United States Food and Drug Administration (FDA) for the treatment of pediatric relapsed/refractory ALL after at least two prior regimens. In adults, clofarabine has shown significant efficacy in hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) alone and in combinations. Ongoing and future studies will examine the use of clofarabine in elderly patients with AML for whom standard regimens are too toxic, and in MDS with intravenous and oral forms of the drug.     

Phenotypic diversity in leukemia cell populations

Summary Acute leukemia comprises a large group of different diseases that can be identified by morphology in combination with immunological markers. Such studies suggest that phenotypic heterogeneity may be expressed in individual leukemia cell populations. This was verified in the murine AKR leukemia that was found to be composed of four antigenically different subtypes of leukemia cells, and it was shown that this feature has a severe negative impact on the use of leukemia cell specific monoclonal antibodies (Mabs) as therapeutical reagents. Twenty-four human T-lymphoblastic leukemias were analyzed with Mabs against HLA class I, HLA class II, and T-lymphocyte differentiation antigens, and 21 were found to be intratumoral heterogeneous with respect to these antigens.Mabs with high specificity were generated against AML cells and subsequently used to analyze more than 50 AML samples from different patients. The reactivity pattern of the Mabs differed significantly among the various AML samples. Further, a pronounced intratumoral antigenic heterogeneity (IAH) was found in most AML samples with regard to reactivity of the Mabs against AML and expression of major histocompatibility antigens.The negative impact of IAH on the use of Mabs in clinical oncology is described. It is argued that IAH examplifies the phenotypic diversity of malignant neoplasms which is also suggested to be a basic and necessary feature of malignant cell populations. Mabs against subsets of malignant cell populations may have a profound effect on cancerous cell populations, and it is therefore of crucial importance that such subsets are identified and characterized. It is conceivable that this may result in generation of Mabs with potentially high value in cancer diagnosis and therapy, particularly in combination with drugs that induce differentiation in the malignant cell mass.     

Cell Surface Antigens of Radiation Leukemia Virus-induced BALB/c Leukemias Defined by Syngeneic Cytotoxic T Lymphocytes

Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus(RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2^d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2K^d, but not H-2D^d. The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines.     

The Role of Decreased Retinoblastoma Protein Expression in Acute Myelomonocytic and Monoblastic Leukemias

The results of different investigators show that lack of p 105 expression is relatively common in human myeloid leukemias, especially in monocytic leukemias. This suggests that loss of p105 expression could contribute to the altered growth control of these cells. So far no clear data exist which show that low p105 levels in AML blasts predict a poor therapy outcome. Therefore it is not very likely that p105 expression will become a strong prognostic factor for the different treatment strategies in AML.     

Oncogenes and leukemia

Cellular or proto-oncogenes are normal cellular genes important in normal cell growth and development. In some instances abnormal expression of these genes is associated with altered cell growth or with malignant transformation. Abnormalities of cellular oncogenes are common in human leukemias. These arise by multiple mechanisms such as mutation, translocation, amplification, and others. Sometimes more than one abnormality is present within a single oncogene. In other instances, a leukemia cell may contain abnormalities of several different oncogenes. Some oncogene abnormalities are relatively specific for certain leukemias and occur in almost all cases; examples include ABL in chronic myelogenous leukemia or MYC in Burkitt leukemia/lymphoma. Other abnormalities are also relatively specific but occur in only some cases such as NRAS in acute myelogenous leukemia or BCL2 in B-cell acute lymphoblastic leukemia. In other leukemias, such as most cases of acute lymphoblastic leukemia and chronic lymphocytic leukemia, oncogene abnormalities are uncommon. The precise role of oncogenes in the pathogenesis of human leukemia is unknown. Retrovirus transduced versions of some of the oncogenes modified in human leukemias cause leukemia in animals. Other oncogenes, modified or unmodified, transform animal and human hematopoietic cells in vitro. Some oncogene products are hematopoietic growth factors or growth factor receptors while others regulate cell proliferation or differentiation by diverse mechanisms. Disruption of the balance between these processes seems the most likely mechanism of oncogene related leukemogenesis. If the role of oncogenes in human leukemias can be defined, innovative diagnostic and therapeutic strategies may be forthcoming.     

Isoenzyme studies in human leukemia -- II. Carboxylic esterase (E.C. 3.1.1.1.)

Isoelectric focusing analysis of esterase activity (E.C. 3.1.1.1.) has been carried out on polyacrylamide gel slabs of a variety of immunologically defined acute leukemia subtypes. Distinct isoenzyme bands and isoenzyme groups have been identified and characterized biochemically with regard to their features for substrate specificity, pH optimum and inhibition experiments. The esterase isoenzyme patterns permitted the distinction between acute myeloid and non-myeloid leukemias. Acute lymphocytic leukemias localized by their immunological profile along the T-cell axis exhibited a readily recognizable isoenzyme group (T group). Reproducible differences in isoenzyme patterns were observed in childhood common ALL which indicate that the immunologically determined entity cALL displays a clear heterogeneity with respect to biochemical profiles of enzyme markers. These results suggest that further combined biochemical and immunological characterization may significantly contribute to a better understanding of lymphopoietic and leukemic cell differentiation.     

DNA ligases in human leukemia

Following partial purification on sucrose gradient and/or phosphocellulose chromatography, DNA ligase was tested in peripheral white blood and bone marrow cells of nearly 100 patients with various kinds of leukemias, mainly acute leukemias. Terminal deoxynucleotidyl transferase (TdT) was tested in parallel. DNA ligase of acute myeloblastic leukemia (AML) was extracted with the same sedimentation coefficient (5.5S) on sucrose gradient, and eluted with the same KCl molarity (0.3 M) than the one extracted from normal lymphocytes. Acute lymphoblastic leukemias (ALL) were characterized by no detectable DNA ligase activity--in most T or non T-non B-ALL, or a low activity in pre-B and B (Burkitt type) ALL, with levels similar to the one observed in chronic lymphocytic leukemia (CLL). An inverse relationship was observed between DNA-ligase and TdT in ALL, ligase being undetectable in cells positive for TdT and being present in some T or non T-non B, and in all pre-B and B-ALL negative for TdT. AML and chronic myelocytic leukemia (CML) were characterized by a markedly higher DNA-ligase activity. This activity was higher in the most differentiated subtypes--M2, M3 and M4 subtypes of FAB classification--and in CML. Moreover a high degree of correlation was observed in AML between the DNA ligase activity and the S phase fraction measured by 3 H-thymidine autoradiography or flow cytophotometry on the total cell sampling. Besides their clinical interest, these results are discussed in relation with the role of DNA-ligase in DNA replication and repair.     

Glutathiones-transferase activity of leukemic cells as a prognostic factor for response to chemotherapy in acute leukemias

This paper presents an analysis of glutathione S-transferase (GST) activity of ieukemic cells in 30 patients with acute leukemias and its predictive value for therapy. Blast cells were isolated from peripheral blood or bone marrow before induction therapy using Ficoll density gradient. GST activity was measured accorcling to the spectrophotometric assay based on the use of l-chloro-2,4-clinitrobenzene as a substrate. The results did not show any significant differences between activities of the enzyme within the different leukemia types accorcling to the French-American-British (FAB) classification. The patients who achieved complete remission demonstrated the lowest value of enzyme activity. The highest enzyme activity was observed in those patients who achieved partial remission and the non-responsive patients presented a GST value within the median of these two groups. Two categories of patients were represented within the non-responsive treatment group. One was resistant to the conventional therapy and in the other death was caused by infectious or hemorrhagic complications. The mean GST activity in these two groups of patients differ greatly. These results suggest that low GST activity of Ieukemic cells could be a favourable prognostic factor whereas high GST values could help to find out the group of patients who should be further analysed prior to induction therapy.     

P2X家族受体在急性T淋巴细胞白血病小鼠中表达的特点

摘 要 目的：探讨不同P2X家族受体在小鼠急性T淋巴细胞白血病发展中的表达变化规律。方法：制备Notch1过表达小鼠GFP+T细胞急性淋巴细胞白血病移植模型,流式术分选CD45.2+GFP+白血病细胞,实时定量PCR检测P2X受体家族的表达变化,荧光分光光度计检测P2X7受体介导的钙离子浓度的变化。结果：对照组和白血病组小鼠骨髓细胞表达除P2X5外的其他6种P2X家族受体。Notch1过表达导致的小鼠白血病发展过程中,P2X7的表达水平逐渐升高,P2X1和P2X3的表达水平逐渐降低,而P2X2、P2X4和P2X6表达水平没有显著变化;分选后的CD45.2+GFP+白血病细胞中,P2X家族受体的表达呈现相同的规律。对照组和白血病组小鼠骨髓细胞中P2X7受体在激动剂苯甲酰-苯甲酸ATP（BzATP）的刺激下都能介导细胞内钙离子浓度升高,但白血病组小鼠骨髓细胞内钙离子处于持续高浓度状态,而对照组小鼠细胞内钙离子浓度短暂升高后逐渐下降;P2X7介导的这种钙离子反应可被其特异的拮抗剂KN62所阻断。结论：P2X受体家族中P2X1、P2X3和P2X7的表达变化与小鼠急性T淋巴细胞白血病的发展相关,提示其介导的细胞间通讯可能在白血病发展中发挥重要作用。     

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins.

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate post-allogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-gamma or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.     

DNA strand breakage and ADP-ribosyl transferase mediated DNA ligation during stimulation of human bone marrow cells by granulocyte-macrophage colony stimulating activity

ADP-ribosyl transferase (ADP-RT) is a chromatin-bound nuclear enzyme catalysing the transfer of ADP-ribose from NAD+ to chromatin proteins. The enzyme is activated by DNA strand breaks and has been suggested to have roles in both DNA repair (via its effect on DNA ligase II) and in differentiation. We recently demonstrated that specific inhibitors of ADP-RT preferentially inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage and that the specific proliferation/differentiation stimulus granulocyte-macrophage colony stimulating activity (GM-CSA) activates ADP-RT in human marrow cells within 3 h of exposure. The purpose of this study was to investigate the role of ADP-RT in monocyte-macrophage differentiation. By altering the time of addition of ADP-RT inhibitor it was demonstrated that maximal inhibition of macrophage differentiation only occurs when the inhibitor is added within the first 24 h of culture. This suggests that it is an early event during the induced differentiation of granulocyte-macrophage progenitor cells which requires ADP-RT. Fluorometric assay of the level of DNA strand breaks showed that GM-CSA induces DNA strand breaks which are rapidly ligated only if ADP-RT is available. These data and those of our earlier studies suggest that DNA rearrangement may be involved in differentiation of granulocyte-macrophage progenitors to the monocyte-macrophage pathway. Such a DNA rearrangement could provide a molecular basis for commitment of multipotent progenitors to a single lineage.     

Isoenzyme studies in human leukemia — III. β-hexosaminidase (E.C. 3.2.1.30)

Enzymologic profiles of β-hexosaminidase (N-acetyl-β-d-glucosaminidase, E.C.3.2.1.30) were studied in cells from childhood acute leukemias and lymphomas. By analytical isoelectric focusing or disc electrophoresis the β-hexosaminidase activity was separated into its components A, B, I and C. The isoenzyme patterns were correlated with immunologic cell surface marker characteristics found on the investigated leukemic cells. In all cases of T-ALL the β-hexosaminidase forms A and B were observed, an enzyme pattern similar to that found in normal lymphocytes. Seven out of 11 cases with cALL, three of six cases with AML and one case of AUL displayed the intermediate component (Hex I). Marked heterogeneity within the immunologically classified subgroup cALL was reflected in different enzyme patterns of the cALL samples. These biochemical phenotypes may indicate the different maturation and differentiation status of cells expressing the same immunologic surface markers.     

Enzymatic differences between chronic lymphocytic leukemia and prolymphocytic leukemia

Chronic lymphocytic leukemia and prolymphocytic leukemia of the B-cell immunophenotype are closely related disorders, but differ in their cytomorphologic and clinical features. In an attempt to differentiate further between these two forms of leukemia, we measured adenosine deaminase and purine nucleoside phosphorylase activities by using a linked-enzyme spectrophotometric assay on peripheral-blood leukemic cells from seven patients with chronic lymphocytic leukemia, three patients with prolymphocytic leukemia, and one patient with prolymphocytoid transformation of chronic lymphocytic leukemia. By using discriminant analysis, we were able to distinguish the two groups only on the basis of purine nucleoside phosphorylase activity (F1,9; p less than 0.001). The purine nucleoside phosphorylase activity in leukemic cells with prolymphocytic cytomorphology was significantly elevated (mean = 58.6 nM/min/mg protein) compared to the activity in leukemic cells with lymphocytic cytomorphology (mean = 25.6 nM/min/mg protein). There was only one patient with chronic lymphocytic leukemia who was assigned to the prolymphocytic leukemia group on the basis of her purine nucleoside phosphorylase activity. Our study suggests that purine nucleoside phosphorylase activity in leukemic cells may be useful in the distinction of prolymphocytic leukemia from chronic lymphocytic leukemia, and that it may be an enzymatic marker for the early detection of prolymphocytoid transformation of chronic lymphocytic leukemia.     

Analysis of human leukemia/lymphoma cell lines with monoclonal antibodies BA-1, BA-2 and BA-3

A panel of monoclonal antibodies (BA-1, BA-2 and BA-3) were made against the pre-B acute lymphoblastic leukemia (ALL) cell line NALM-6. BA-3 binds to the 100,000 mol. wt common ALL antigen (gp100/CALLA), BA-2 binds to a 24,000 mol. wt (p24) cell surface structure and BA-1 binds to a currently undefined antigen. These antibodies were analysed for their reactivity with 67 human leukemia/lymphoma cell lines. The cell lines studied included T ALL, non-T ALL. surface immunoglobulin⁺ leukemias/lymphomas, and myeloid/monocytoid leukemias. The results indicate that all three antibodies react primarily (but not exclusively) with malignant cells early in B cell development. This pattern of reactivity was similar to previous results obtained with fresh, non-cultured malignant cells. BA-1, BA-2 and BA-3 are useful tools for analysing the developmental options of normal lymphoid cells and for “cleaning up” leukemia/lymphoma cells in selected cases of autologous bone marrow transplantation.