[Show abstract][Hide abstract] ABSTRACT: Apoptosis is a prominent mechanism of programmed cell death in the immune system. In the thymus apoptosis is responsible for the deletion of autoreactive T-cells during thymic differentiation. The typical features of apoptosis are characterized by nuclear and cytoplasmic morphologic changes, along with cleavage of chromatin at regularly spaced sites. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerizing enzyme found at an early stage of T and B lymphocyte differentiation, which generates diversity in the DNA sequence of immunoglobulin (Ig) or T cell receptor (TCR). The combined evaluations of thymocyte morphological features, immune phenotype and thymic topography associated to TdT expression allow the recognition of three different thymocyte subpopulations, characterized by small-size, intermediate-size and large-size. The results of this study show that dexamethasone (Dx)-treatment induces cell death via apoptosis involving distinct transformations related to differentiation stages of thymic subpopulations. Intermediate and small-size thymocytes that are TdT-negative or weakly positive at nuclear level are Dx sensitive. In contrast the large-size thymocytes, highly TdT positive, corresponding to the undifferentiated cells, do not show significant morphological modifications and TdT positivity to Dx-treatment. Immunocytochemical analysis shows that Dx-treatment does not affect TdT synthesis but morphological changes, occurring during apoptotic process, are responsive to intracellular movement and intranuclear arrangement of the TdT.
Cell Structure and Function 01/1996; 20(6):455-63. DOI:10.1247/csf.20.455 · 1.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Terminal deoxynucleotidyl Transferase (TdT) play an essential role in the immune system differentiation. KM-3 cells are lymphoblastoid cells expressing the TdT and when induced to differentiate by phorbol ester (PMA) they loose this enzyme. Therefore, because of the suggested involvement of polyphosphoinositide in controlling the nuclear events it has been analyzed the phosphorylation of nuclear polyphosphoinositides during KM-3 differentiation. When the differentiated state is reached the phosphorylation level of PIP2 increases in isolated nuclei and this is accompanied by a concomitant decrease of PIP and PA, hinting at a correlation between polyphosphoinositide metabolism and TdT expression.
Biochemistry and molecular biology international 05/1993; 29(6):1123-30.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The ability to overproduce terminal transferase through recombinant DNA technology should provide alternate means for generating sufficient quantities for structural and mechanistic study of this creative DNA polymerase. In this work we have investigated, at electron microscope level, the morphological modification and ultrastructural localization of synthesized human terminal transferase occurring in Sf-9 cells during recombinant baculovirus infection time. The results obtained showed that TdT is localized and stored only at the cytoplasmic level; the nucleus did not show any specific site able to link the neosynthesized TdT. The amount of the enzyme, estimate by immunostaining analysis, increased with the viral infection time. Morphological changes occurring during viral infection consist mainly of variations of cellular surface, different size and shape of cytoplasmic organelles and modification of nuclear components.
Cell Structure and Function 11/1992; 17(5):287-92. DOI:10.1247/csf.17.287 · 1.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.
Experimental Cell Research 11/1992; 202(2):405-11. DOI:10.1016/0014-4827(92)90093-N · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TdT positive cells in rat thymus belong to distinct subsets as shown by light and electron microscopic immunocytochemistry. Using polyclonal antibodies to calf TdT and peroxidase labeled goat anti-rabbit IgG it has been possible to identify several subpopulations of TdT positive thymocytes. Large blasts corresponding to thymocytes at early maturational stages, are strongly positive for TdT which is diffusely distributed in both the nucleus and the cytoplasm. Smaller cells which correspond presumably to more advanced stages of maturation display nuclear TdT only, or are negative. Ultrastructural analyses of TdT indicate that the localization of the enzyme is related to the morphological features of the cells and TdT expression corresponds to maturational stages of T-cells.
[Show abstract][Hide abstract] ABSTRACT: Nuclear matrix extracted from KM-3, a human pre-B leukemia cell line, appears to have a site of linkage for terminal deoxynucleotidyl transferase (TdT). The immunocytochemical analysis of the distribution of TdT using a rabbit polyclonal antibody which recognizes human terminal transferase, shows that the nuclear framework of these cells contains sites of immunoreactivity that appear uniformly distributed on the matrix fibres, while the nucleolar region is unreactive. This evidence points out the possibility that TdT could reside in the proteinaceous scaffold of the nucleus defined as nuclear matrix, thus strengthening the evidence for the metabolic and regulatory roles ascribed to this nuclear framework.
[Show abstract][Hide abstract] ABSTRACT: Terminal deoxynucleotidyltransferase activity is absolutely dependent on the presence of a divalent cation in the reaction mixture. This requirement can be satisfied by either Mg2+, Co2+, or Mn2+. When Mg2+ is used, the reaction rate is inhibited by metal ligands, and this inhibition can be reversed by Zn2+. Reaction rates in Mg2+ are also stimulated by the addition of micromolar amounts of Zn2+. To examine the role of Zn2+ in terminal transferase catalysis we analyzed for Zn2+ in homogeneous recombinant human terminal transferase preparations and found that Zn2+ is not an intrinsic part of enzyme molecule. Analysis of Zn2+ binding to terminal transferase under equilibrium conditions shows about 0.3 g of atom of Zn2+/mol of enzyme, suggesting that Zn2+ forms an easily dissociable complex with the enzyme molecule. Kinetic analyses showed that the stimulatory effect of Zn2+ is observed in several buffer systems. Zn2+ increases the affinity of the enzyme for the initiator about 2-fold and decreases affinity for dATP more than 10-fold, resulting in an increase in the apparent Vmax of the reaction. Using a 3'-ended 2',3'-dideoxyoligonucleotide as an inhibitor demonstrates that the inhibitor has no effect on the reaction rate in the absence of Zn2+ but is competitive with respect to the initiator in the presence of Zn2+. These results suggest that Zn2+ is a positive effector for terminal transferase, interacting with oligonucleotide and enzyme near the initiator binding site. Binding of Zn2+ to the enzyme appears to induce conformational changes that greatly increase the Vmax of the reaction with a concomitant decrease in the affinity of the enzyme for dNTP.
Journal of Biological Chemistry 11/1990; 265(29):17436-40. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Leukemia Research 02/1990; 14(3):273-8. DOI:10.1016/0145-2126(90)90135-V · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have established stably transformed mammalian cell lines expressing recombinant human terminal deoxynucleotidyl transferase. A 58 kDa, enzymatically active protein is produced by these cell lines. Using the lacI gene of pJYMib shuttle vector as mutagenic target, we found no increase in mutation rates in cells expressing terminal deoxynucleotidyl transferase compared to controls. Our results suggest that the presence of terminal deoxynucleotidyl transferase alone in mammalian cells does not increase mutation rates.
Biochemical and Biophysical Research Communications 12/1989; 165(1):271-7. DOI:10.1016/0006-291X(89)91065-6 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.
Journal of Biological Chemistry 10/1988; 263(25):12509-13. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Philadelphia chromosome-positive (Ph1) acute leukemia is a heterogeneous subset of acute leukemia with a poor prognosis. We studied five patients to determine the potential for phenotypic and molecular heterogeneity. Cellular characterization studies included light myeloperoxidase (L-MPO), terminal deoxynucleotidyl transferase (TdT), ultrastructural MPO (U-MPO), and immunophenotyping by flow cytometry using T11, T3, T4, T8, Leu 1, B1, Leu 12, HLA-DR (la), CALLA (J5), OKM1, My4, My7, My8, My9, and My10. DNA was analyzed for rearrangements of the breakpoint cluster region (bcr), immunoglobulin heavy chain, joining region (JH), immunoglobulin kappa light chain constant region (C kappa), and T cell receptor (TcR beta). RNA dot blots were hybridized by using molecular probes for MPO and TdT. We found that four of five cases were acute mixed-lineage leukemia (AMLL). One patient had acute unclassifiable leukemia. Of the four patients classified as having AMLL, three showed myeloid and lymphoid features, with one patient showing myeloid, T cell, and B cell features. The last case showed T cell and B cell features only. In one patient MPO/RNA was positive in spite of insufficient L-MPO or U-MPO to diagnose acute myelogenous leukemia (AML), thereby suggesting significant MPO gene expression before the production of sufficient MPO protein to meet the French-American-British criteria for AML. Three of the five patients showed rearrangement of bcr (cases 1, 2, and 5). Studies of these five patients support the concepts of molecular and phenotypic heterogeneity in Ph1 acute leukemia, demonstrate a high incidence of AMLL in this subset of acute leukemia, and support the use of lineage-associated molecular probes to define lineage at an earlier stage than previously possible.
[Show abstract][Hide abstract] ABSTRACT: We have localized a cDNA fragment that codes for human DNA polymerase-beta. Using somatic cell and in situ hybridization techniques, this cDNA was cloned by screening a human KM-3 cell cDNA library in lambda gt 11 for expression of fused beta-galactosidase-human DNA polymerase-beta proteins. We have mapped this human polymerase-beta gene to the short arm of chromosome 8 in the subregion 8p11----p12.
Cytogenetics and cell genetics 02/1988; 47(3):121-4.
[Show abstract][Hide abstract] ABSTRACT: Methods for using rabbit polyclonal and mouse monoclonal antibodies to detect terminal deoxynucleotidyl transferase (TdT) in human leukemia lines were developed. Use of 1% paraformaldehyde in 0.1 M Na cacodylate buffer, pH 7.6, as a fixative provides a procedure that can be used for immunofluorescence, immunoalkaline phosphatase (APAAP), or horseradish peroxidase conjugate detection procedures in these cell lines. The peroxidase procedure can also be used for electron microscopy, clearly demonstrating nuclear localization of TdT in interphase nuclei of TdT-positive cells.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Experimental Medicine 11/1986; 164(4):1129-44. · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.
Journal of Biological Chemistry 10/1986; 261(25):11924-30. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal of Clinical Pathology 08/1986; 86(1):88-91. · 2.51 Impact Factor