Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. II. Isolation of terminal deoxynucleotidyl transferase-positive cells.

A method is described by which highly enriched populations of viable terminal deoxynucleotidyl transferase-positive (TdT+) cells can be isolated from rat bone marrow by use of the fluorescence-activated cell sorter. Such cells have been postulated to be progenitors of thymocytes and, possibly, of B lymphocytes, and may serve as the targets of neoplastic transformation in acute lymphoblastic leukemia. The separation procedure is based on differences in relative low-angle light scatter and relative fluorescence intensity for Thy-1 antigen between TdT+ cells and other lymphohemopoietic cell populations in bone marrow. Simultaneous sorting of bone marrow cells according to these two parameters resulted in a mean 87% purification of TdT+ cells. The morphological characteristics of the isolated TdT+ cells are described at the light and electron miscroscopic levels.

Defective lymphopoiesis in bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. I. Analysis of development of prothymocytes, early B lineage cells, and terminal deoxynucleotidyl transferase-positive cells.

This study identifies defects in the early stages of lymphopoiesis that may contribute to the abnormalities in the development and/or function of peripheral T and B lymphocytes in mice homozygous for the motheaten (me/me) and viable motheaten (mev/mev) mutations. The results indicate that in me/me and mev/mev mice prothymocytes in bone marrow are present in essentially normal numbers, as determined by intrathymic injection, but apparently lack the ability to home effectively to the thymus, as determined by intravenous transfer; early B lineage cells in bone marrow, identified by the B220 antigen, are markedly depleted, including immature B cells (sIg+), pre-B cells (cIg+, sIg-), and pro-B cells (B220+, cIg-, sIg-); TdT+ bone marrow cells, especially a subset that expresses the B220 B lineage antigen, are markedly depleted by two weeks of age; normal numbers of TdT+ thymocytes are present during the first 3 wk of postnatal life, but rapidly decrease thereafter. The results further indicate that neither the defective thymus homing capacity of prothymocytes nor the deficiency of TdT+ bone marrow cells is due to autoantibodies. The possible relationship of the defective development of lymphoid precursor cells to the premature onset of thymic involution and to the abnormalities of peripheral T and B lymphocytes in me/me and mev/mev mice is discussed; as are the results of in vitro studies (presented in a companion paper), which suggest that a primary defect in the stromal microenvironment of the bone marrow is responsible for the abnormal development of the lymphoid precursor cells.

Incorporation of nucleotides into nuclei of fixed cells by DNA polymerase.

The enzyme calf thymus polymerase requires denatured or single-stranded DNA as a primer for DNA synthesis and is inactive on native DNA preparations. The enzyme and tritium-labeled deoxyribonucleoside triphosphates were incubated with alcohol-fixed and Carnoy-fixed tissue preparations to see if primer DNA could be found in several types of cells undergoing DNA synthesis. In all cases, low-pH controls were prepared for comparison. Priming activity was not found in nuclei that had been fixed in alcohol. Priming activity was found in cell nuclei that had been fixed with an acid fixative or had been treated at a low pH prior to treatment with the enzyme reaction mixture.

Antigenic relationships in mammalian DNA polymerase.

Rabbit antibody was prepared against a high-molecular-weight DNA polymerase purified from the soluble fraction of calf thymus gland. This antibody does not inhibit terminal deoxynucleotidyl transferase isolated from that source, but does inhibit both low-molecular-weight and high-molecular-weight DNA polymerases isolated from cytoplasmic and nuclear fractions of a number of mammalian tissues (mouse L cells, calf thymus, phytohemagglutinin-stimulated human lymphocytes, rat liver, and rabbit bone marrow). The results suggest that (i) no antigenic relationship exists between terminal transferase and DNA polymerase, (ii) common antigenic determinants exist in the DNA polymerases from all mammalian sources, and (iii) multiple forms of DNA polymerase found in mammalian, cells are related by having polypeptide sequences or subunits in common.

Pyrophosphorolytic dismutation of oligodeoxy-nucleotides by terminal deoxynucleotidyltransferase.

Terminal transferase (TdT), when incubated with a purified(32)P-5"-end-labeled oligonucleotide of defined length in the presence of Co(2+), Mn(2+)or Mg(2+)and 2-mercaptoethanol in cacodylate or HEPES buffer, pH 7.2, exhibits the ability to remove a 3"-nucleotide from one oligonucleotide and add it to the 3"-end of another. When analyzed by urea-PAGE, this activity is observed as a disproportionation of the starting oligonucleotide into a ladder of shorter and longer oligonucleotides distributed around the starting material. Optimal metal ion concentration is 1-2 mM. All three metal ions support this activity with Co(2+)> Mn(2+) congruent with Mg(2+). Oligonucleotides p(dT) and p(dA) are more efficient substrates than p(dG) and p(dC) because the latter may form secondary structures. The dismutase activity is significant even in the presence of dNTP concentrations comparable to those that exist in the nucleus during the G(1)phase of the cell cycle. Using BetaScope image analysis the rate of pyrophosphorolytic dismutase activity was found to be only moderately slower than the poly-merization activity. These results may help explain the GC-richness of immunoglobulin gene segment joins (N regions) and the loss of bases that occur during gene rearrangements in pre-B and pre-T cells.

Subpopulations of normal and leukemic human thymocytes: an analysis with the use of monoclonal antibodies.

Combinations of antibodies to membrane antigens and to terminal deoxynucleotidyl transferase (TdT) were used to study human thymocyte and bone marrow subpopulations and leukemia cells. Cortical thymocytes were TdT+ and expressed T-cell antigens (HuTLA+), a thymocyte-specific antigen (HTA-1+), and a leukocyte antigen (HLe-l++) but lacked detectable HLA-A,B,C and la (HLA-D) antigens. In contrast, medullary thymocytes were TdT-, HuTLA+, HTA-1-, HLe-l++. A small subpopulation of larger, probably immature, thymocytes were strongly TdT+, HuTLA+, la-, HTA-1-, HLe-l +/-. Many blast cells from cases of thymic acute lymphoblastic leukemia (Thy-ALL) showed the phenotype of this small subset, and only a proportion of Thy-ALL blast cells exhibited HTA-1 and HLe-l antigens as strongly as was observed on normal cortical thymocytes. In contrast, TdT+ cells observed in normal juvenile bone marrow were HuTLA, HTA-1-, HLA+, la+. This phenotype corresponded to the phenotype of the common form of ALL (non-T, non-B) and indicated that further studies are necessary to analyze the differentiation of bone marrow precursors to thymic cells.

Serial observations on terminal deoxynucleotidyl transferase activity and lymphoblast surface markers in acute lymphoblastic leukemia.

Terminal deoxynucleotidyl transferase activity and cell surface markers were measured in peripheral lymphoid cells from 27 children with acute lymphoblastic leukemia in various phases of their disease. Lymphoblasts from untreated patients had smooth surface ultrastructure but heterogeneous surface receptors. Greater than 60% of lymphoblasts from 4 to 7 untreated patients formed rosettes with sheep red blood cells. Transferase activity was variable, ranging from 8 to 210 units/10(8) blasts, but it was consistently elevated at diagnosis and in relapse. Transferase levels did not correlate with the presence of lymphoblast surface receptors. During induction therapy transferase activity decreased rapidly, but it remained elevated in peripheral lymphoid cells even when blasts were not detectable in peripheral blood smears. Patients in remission had normal surface receptors and undetectable or minimally elevated levels of transferase. Terminal transferase activity may be a sensitive biochemical marker for a primitive cell population and may be important in the evaluation of therapeutic effectiveness in acute lymphoblastic leukemia.

Clinical utility of initial terminal deoxynucleotidyl transferase determinations in childhood acute leukemias.

Terminal deoxynucleotidyl transferase (TDT) activity was measured in bone marrow lymphoblasts obtained at diagnosis from 168 consecutive patients with childhood acute leukemia. Absolute concentrations of TDT were increased (greater than or equal to 20 units/10(8) blasts) in samples from 98 of 112 assessable patients with acute lymphocyte leukemia (ALL). The values ranged from less than 1 to 1502 units/10(8) blasts with a median of 90 units contrasted with less than 1 to 219 units (median, 2.6 units) in studies of children without leukemia. Results of an immunofluorescence assay were in good agreement with enzymatic detection of the polymerase. Among 115 patients with adequate marrow smears, 105 had TDT-positive blasts. By contrast, in most children with acute myelogenous leukemia, TDT activity was either undetectable or less than 10 units/10(8) blasts. Although the highest levels of TDT were found in blasts with the common ALL phenotype, quantitative determinations were not significantly related to the major immunological subtypes of ALL or to morphological features or periodic acid-Schiff reactivity of the lymphoblasts. The probability that a newly diagnosed case of leukemia would be ALL was 90% if TDT levels were greater than 20 units/10(8) blasts. We conclude that absolute concentrations of TDT, as determined in this study, are of little value in identifying subclasses of ALL. The immunofluorescence assay, which is much less expensive and easier to perform than the enzyme assay, should prove useful for confirming the diagnosis of ALL and for detecting extramedullary sites of leukemic infiltration.

DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients.

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.

Sensitivity and applicability of different methods for detection of terminal transferase in leukemia.

The sensitivity of terminal deoxynucleotidyl transferase (TdT) assay methods was examined by using a mixture of the TdT-positive lymphoblastic leukemia cell line NALM-18 and the TdT-negative erythroleukemia cell line K-562. The biochemical assay could detect TdT activity in the mixture containing NALM-18 cells at concentrations of more than 10 percent. The immunofluorescent (IF) method could detect positive cells in the mixture containing NALM-18 cells at concentrations of more than 1 percent. Furthermore, an approximately 10(5)-fold increase in sensitivity was obtained by the combination of RT-PCR and subsequent Southern blotting, as compared to biochemical assay. In many leukemia cases the expression of TdT-mRNA corresponded well to that of TdT protein. However, in some patients with leukemia, only TdT-mRNA was detectable by RT-PCR without any expression of TdT protein. A PCR-based technique enables us to detect TdT transcripts at the highest sensitivity, but does not allow the characterization of each positive cell. IF analysis is simple and sensitive, but may sometimes cause nonspecific reactions. All these techniques have some advantages and some faults, therefore, the results obtained from clinical studies using these techniques should be interpreted with caution.

Ontogenic studies of lymphoid progenitor cells in human bone marrow.

The enzyme terminal deoxynucleotidyl transferase (TdT) is considered a useful marker of human bone marrow lymphoid progenitor cells (i.e., those cells that lack surface immunoglobulin and sheep erythrocyte receptors). The cell surface molecule p24 (identified by monoclonal antibody BA-2) has also been identified on a significant number of bone marrow lymphoid progenitor cells. The current study was undertaken to analyze adult, pediatric, and fetal bone marrow lymphoid cells for the expression of p24 and TdT. We document, for the first time, that fetal bone marrow represents a highly enriched source of TdT+ cells. We also show that dramatic quantitative differences exist in the percentage of p24+ and TdT+ cells from human bone marrow at different stages of ontogeny, with highest numbers in fetal marrow, intermediate numbers in pediatric marrow, and lowest numbers in adult marrow. Such differences are consistent with the hypothesis that marrow lymphopoiesis is an active process early in ontogeny that decreases with age.

Terminal deoxynucleotidil transferase is a nuclear PKC substrate.

Protein phosphorylation is the regulatory mechanism of many cellular events in response to changes in metabolic activity and environmental conditions. Seeing that PKC and TdT levels in cells are both regulated by PMA, we sought particularly intriguing to investigate TdT phosphorylation in vivo, utilizing KM-3 cells, a TdT-positive human pre-B cell line treated with PMA and in vitro, employing purified PKC and human recombinant TdT. Our data show that TdT is a substrate for PKC activity, suggesting that TdT phosphorylation could play a key role in the pathway affecting the control of gene transcription and protein synthesis during lymphoid cells differentiation.

Phorbol ester-induced effects on cell cycle progression and terminal deoxynucleotidyltransferase (TdT) activity in KM-3 pre-B cell line.

Phorbol myristic acetate (PMA) is a tumor-promoting agent that has been shown to induce differentiation of human leukemia cells and of normal lymphoid cells. We have investigated the ability of PMA to induce inhibition of cell growth of the human KM-3 pre-B leukemic cell line by multiparametric analysis. Our results show that PMA treatment induces cell differentiation with the disappearance of terminal deoxynucleotidyltransferase and a decrease of cell growth, as evaluated by [3H]thymidine uptake. Flow cytometric analysis of BrdU incorporation shows that PMA is able to induce a modification of the cell cycle with a sharp decrease of the percentage of S-phase cells, which is more evident after 24 h of treatment. Comparison between the cell growth kinetics and TdT synthesis and activity shows that differentiated cells are still able to proliferate to a certain extent and that the TdT disappearance and the initial decrease of cell proliferation are two independent effects of PMA.

Sialyltransferase activity in leukemia.

Sialyltransferase activity in blasts from acute lymphoblastic leukemia (ALL) was markedly lower (1.68 +/- 1.23 pmol/mg protein) than those (6.18 +/- 2.22 pmol/mg protein) of lymphocytes from normal volunteers (t less than 0.001). On the contrary, enzyme activity was significantly increased in blasts (1.21 +/- 0.38 pmol/mg protein) from acute non-lymphoblastic leukemia, compared to the level (0.53 +/- 0.32 pmol/mg protein) of mature granulocytes (t less than 0.001). In TdT-negative CML in blast crisis, sialytransferase activity (2.11 +/- 0.88 pmol/mg protein) was significantly higher than those of mature granulocytes (t less than 0.001), whereas no significant difference in the enzyme activity was noted between the blasts from TdT-positive CML in blast crisis and from ALL. In TdT-positive ALL cases, there was an inverse relationship (r = -0.85, t less than 0.01) between sialytransferase activity and terminal deoxynucleotidyl transferase (TdT) activity of the blasts. Therefore, sialytransferase in leukocytes may be a unique enzyme in which changes in activity relate to the differentiation or malignant transformation of leukocytes.

Polyadenylic acid polymerase activity in chronic myelogenous leukemia.

Poly(A) polymerase activity was markedly elevated in CML in the blastic phase, moderately high in the accelerated phase and low in the chronic phase. The activity was significantly higher in the myeloid crisis than in the lymphoid crisis and elevated with increasing ratio of blasts in leukemia cases. In TPA or retinoic acid-treated leukemia cells poly(A) polymerase activity was decreased. These results suggest that poly(A) polymerase activity changes, depending on the maturation of leukemic cells and the assay of this enzyme activity may be useful for the early detection of the exacerbation of CML cases.

Detection of terminal deoxynucleotidyl transferase in acute leukemias using monoclonal antibodies directed against native and denatured sites.

Monoclonal antibodies (MoAb) directed against human terminal deoxynucleotidyl transferase (TdT) have been developed recently. The authors evaluated the reactivity of two TdT MoAb, one directed against a native site and the other against a denatured site, in bone marrow samples from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). Results were correlated with the immunophenotype and compared with those obtained with an anti-TdT polyclonal antibody (PoAb). The authors found that 39 of the 45 children (87%) with ALL were positive with the anti-TdT PoAb, while only 25 of 45 (56%) were positive using MoAb. In the 41 of 45 cases of ALL for which marker studies were available, there was no relationship between immunophenotype and reactivity with the PoAb or either MoAb. Five cases of AML were studied and two were positive using the PoAb, but none showed staining with the MoAb. The authors' findings demonstrate that, although MoAb may be used to detect TdT in acute leukemia, the two MoAb used do not correlate with immunophenotype and are less sensitive than the PoAb. However, the MoAb appears to demonstrate more specificity for ALL than the PoAb, since it was not reactive in PoAb+ AML.

Determination of terminal deoxynucleotidyl transferase on bone marrow smears by immunoperoxidase.

Terminal deoxynucleotidyl transferase (TdT) is a marker of lymphoblastic disorders. Previously, the technique for determining TdT on smears was by indirect immunofluorescence. An immunoperoxidase procedure for detecting TdT on smears is reported. Results are in a comparable range with the indirect immunofluorescent technique. As discussed in this paper, TdT by immunoperoxidase may offer certain advantages over the indirect immunofluorescent procedure.

Unusual immunofluorescence patterns for terminal deoxynucleotidyl transferase in blast crisis chronic myelogenous leukemia.

An unusual cytoplasmic distribution of terminal deoxynucleotidyl transferase (TdT) antigen in leukemic cells from two patients who had chronic myelogenous leukemia in blastic phase is described. In most leukemic cells that contain TdT, the intracellular location has been reported to be exclusively nuclear. The cells from these two patients demonstrated TdT staining in both the nucleus and the cytoplasm. The pattern is remarkably similar to that observed in thymocytes, in which bright cytoplasmic staining may also be seen. In the immunofluorescence procedures for detection of TdT in blasts from patients who have chronic myelogenous leukemia, significant cytoplasmic staining should not be mistaken for nonspecific absorption of immunoglobulins or specimen deterioration.

Terminal deoxynucleotidyl transferase immunofluorescence of bone marrow smears: experience in 156 cases.

Terminal deoxynucleotidyl transferase (TdT) is a valuable marker for non-B-, non-T-, and T-cell lymphoblastic disorders. Determination of TdT activity by enzymatic assay is laborious and requires fresh cells. The authors evaluated a new technic for TdT determination, using indirect immunofluorescence on air-dried bone marrow smears. Specimens from 156 consecutive patients with hematologic, oncologic, and other disease states were tested. The TdT visualization by indirect immunofluorescence gives results comparable to those previously reported for lymphoproliferative disorders. Several unusual cases with positive TdT were uncovered. TdT by indirect immunofluorescence is a convenient and rapid technic that enables easier access to TdT determinations than the enzymatic assay.

Terminal transferase in acute lymphoblast leukemia in remission.

Terminal deoxynucleotidyl transferase (TdT) is a marker for the diagnosis of acute lymphoblastic leukemia. To determine its value as an indicator of bone marrow remission or impending relapse, serial remission marrows (less than 5% blasts) from 49 patients who had acute lymphoblastic leukemia were examined for TdT by immunofluorescence over the period of a year. Thirty-eight patients (78%) had less than 1% TdT-positive cells. Eleven patients (22%) had slightly elevated levels of TdT (2%-7%) at some time during the study. of these eleven, only two had relapses; however, neither patient had greater than 1% TdT-positive cells within the three months before the relapse. Therefore, it appears that the presence of slightly increased numbers of TdT-positive cells in acute lymphoblastic leukemia remission bone marrows (2%-7%) does not denote impending relapse. In addition, most of the patients with acute lymphoblastic leukemia in remission had less than 1% TdT-positive cells.