Identification of a C-rich element as a novel cytoplasmic polyadenylation element in Xenopus embryos.

During Xenopus early development, the length of the poly(A) tail of maternal mRNAs is a key element of translational control. Several sequence elements (cytoplasmic polyadenylation elements) localized in 3' untranslated regions have been shown to be responsible for the cytoplasmic polyadenylation of certain maternal mRNAs. Here, we demonstrate that the mRNA encoding the catalytic subunit of phosphatase 2A is polyadenylated after fertilization of Xenopus eggs. This polyadenylation is mediated by the additive effects of two cis elements, one being similar to already described cytoplasmic polyadenylation elements and the other consisting of a polycytosine motif. Finally, a candidate specificity factor for polycytosine-mediated cytoplasmic polyadenylation has been purified and identified as the Xenopus homologue of human alpha-CP2.

Differential regulation of the translation and the stability of two maternal transcripts in preimplantation rabbit embryos.

In most species, transcription is essentially silent during the first mitotic cell cycles that follow fertilization. This means that the regulation of gene expression in early embryos heavily relies on the translational activation or inactivation of maternal mRNAs. In mammals, the mechanisms that control the translation of maternal mRNAs have been mainly studied in the mouse when maternal to zygotic transition occurs after the first mitotic division. In other mammalian species, however, this transition occurs later after several cell cycles, and little is known concerning the regulation of maternal information during this period. To address this question, we have used rabbit pre-implantation embryos to analyze the translational activation and stability of two maternal mRNAs, mm 41 and mm61. During the cleavage period, these mRNAs exhibit distinct kinetics for both their translational activation and degradation. In addition, these mRNAs both undergo cytoplasmic polyadenylation but with different efficiencies. This polyadenylation was functionally correlated with the translational activation of these mRNAs; inhibiting polyadenylation prevented translational activation. The differential efficiency of cytoplasmic polyadenylation, driven by cis-elements in the 3' untranslated region of these mRNAs, was also observed in Xenopus laevis embryos, which emphasizes the high conservation of this mechanism between species.

EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos.

During Xenopus early development, gene expression is regulated mainly at the translational level by the length of the poly(A) tail of mRNAs. The Eg family and c-mos maternal mRNAs are deadenylated rapidly and translationally repressed after fertilization. Here, we characterize a short sequence element (EDEN) responsible for the rapid deadenylation of Eg5 mRNA. Determining the core EDEN sequence permitted us to localize the c-mos EDEN sequence. The c-mos EDEN confered a rapid deadenylation to a reporter gene. The EDEN-specific RNA-binding protein (EDEN-BP) was purified and a cDNA obtained. EDEN-BP is highly homologous to a human protein possibly involved in myotonic dystrophy. Immunodepleting EDEN-BP from an egg extract totally abolished the EDEN-mediated deadenylation activity, but did not affect the default deadenylation activity. Therefore, EDEN-BP constitutes the first trans-acting factor for which an essential role in the specificity of mRNA deadenylation has been directly demonstrated.

An appraisal of the developmental importance of polyamine changes in early Xenopus embryos.

The biological importance of the various changes in polyamine metabolism that occur during early Xenopus development have been investigated. Incubation of embryos in high salt medium was observed to cause a precocious fall in ornithine decarboxylase activity without affecting development. Similarly, inhibiting ornithine decarboxylase activity with specific inhibitors did not affect development. Injecting spermidine, within physiologically relevant limits, caused a dose-dependent inhibition of mitotic divisions in the injected blastomere. Increasing the intracellular putrescine did not affect cell division or development. Co-injection of both spermidine and putrescine, so that the original molar ratio of these two polyamines was conserved, abrogated the inhibition of cell division observed when spermidine was injected alone. Therefore, in Xenopus embryos the intracellular spermidine concentration must be retained within certain limits relative to that of putrescine to allow normal development.

A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines.

The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.

Effects of clinical combinations of antileukemic drugs on DNA ligase from human thymocytes and normal, stimulated, or leukemic lymphocytes.

Human DNA ligase was purified from different kinds of immunocompetent cells: thymocytes, normal and stimulated lymphocytes, blasts from ALL (Burkitt and non-T, non-B) and ANLL (M1, M2, and M5). Based upon the protocol for the treatment of these leukemias, the purified enzymes were assayed in the presence of routinely used combinations of antileukemic drugs. At the range of concentration tested (between 0.1 and 5 microM) some drugs taken separately were totally inactive on the enzyme from the different sources. For those being inhibitory, when used in combination their effect was always different from what was observed when the compound was tested alone. Some combinations were more effective in inhibiting the enzyme from leukemic than from normal cells (vincristine + cyclophosphamide + prednisone in ALL and rubidazone + Ara-C, Ara-C + m-AMSA, in ANLL). However, some combinations of drugs are without effect on ligase from leukemic cells at this dose range (vincristine + rubidazone + Ara-C + prednisone and adriamycin + asparaginase + Ara-C in ALL or etoposide + Ara-C, adriamycin + cyclophosphamide in ANLL). This is the first direct observation of the effect of cytostatic drugs on DNA ligase, a key enzyme of the DNA replication and repair process. The clinical consequences of these observations are discussed in an attempt to selectively inhibit replication, thereby division, of cancer cells.

[Enzymes involved in the metabolism, replication and repair of DNA in acute leukemias (DNA ligases)]. / Enzymes impliquées dans le métabolisme, la replication et la réparation du DNA dans les leucémies aiguës (DNA ligases).

DNA ligases are involved in DNA replication, repair and recombination. Consecutively to partial purification, these enzymes have been studied in acute leukemias and subclasses. There is a good correlation between this enzyme activity and the percentage of cells in S phase in acute myeloblastic leukemia. However, in acute lymphoblastic leukemia, a low and even absent activity (T-ALL) is observed. It is shown that in this type of leukemia, the absence of activity is due to either the absence or the non expression of the DNA ligase gene. The results are discussed in terms of the correlation between the absence of ligase activity and the expression of the TdT phenotype.

Changes in the catalytic properties of DNA ligases during early sea urchin development.

Two distinct DNA ligases are expressed during early sea urchin embryogenesis. A light form (50 kDa) is found in unfertilized eggs (oocyte form) and a heavier enzyme (110 kDa) is observed at the two-cell stage (embryonic form). The chronology of the change reveals that the embryonic form is detected 90 min after fertilization. After the two proteins were purified, their catalytic properties were studied using different substrates. The oocyte ligase acts only on deoxypolymers while the embryonic form also ligates heteropolymers. The two enzymes were found to undergo both nick and cohesive-end ligation. With different kinds of restriction sites it was observed that the embryonic enzyme could also ligate blunt-ended DNA. These catalytic properties account for sealing of exogenous DNA and concatenation following DNA injection into eggs. The role of the oocyte form of the enzyme is unclear; one speculation is a role in repair of DNA breaks which might accumulate during long-term sperm and oocyte storage in the gonad.

Differentiation of thymocytes during human ontogeny: stage-specific DNA ligase in relation to terminal deoxynucleotidyl transferase, cell size and surface antigen.

The activities of two forms (7.5 and 5.5 S) of DNA ligase and of terminal deoxynucleotidyl transferase (TdT) have been studied in human thymocytes at different ages from 20 weeks pre-natal to 37 years after birth. Thymocytes have been selected on the basis of relative size and antigenicity (CD3, OKT3 immunofluorescence) with the cell sorter. For DNA ligases, three kinds of cells can be distinguished: (i) large antigenically negative cells of 20-week fetus, expressing only the 7.5 S enzyme; (ii) large antigenically positive cells without ligase activity; (iii) smaller antigenically positive cells, expressing only the 5.5 S enzyme. This last form of enzyme is found after birth. With respect to TdT expressed in OKT3- 5 micron cells and to OKT3+ thymocytes, it is observed that 5.5 S DNA ligase is found in a thymocyte population distinct from cells expressing TdT. Therefore, these results allow us to consider the 5.5 S DNA ligase activity as an additional functional marker for thymocyte maturation in humans.

Relationship between thymus ontogeny and DNA ligase expression in the chicken.

DNA ligase expression was studied in the embryonic chicken thymus. As previously reported, during thymus ontogeny two separate and successive (8S and 6S) forms of enzyme are found. The switch from the heavy to the light form takes place at around 18 days of incubation in situ. The demonstration that two successive generations of thymic lymphoid precursor cells develop in the embryonic bird thymus and that the replacement of the first by the second thymocyte generation takes place at around 18 days suggested the possibility that each form of enzyme could be restricted to a given thymocyte generation. By either culturing or transplanting embryonic thymuses of different ages it appears that the switch from the heavy to the light form of DNA ligase is not related to a change over of the first by the second generation of embryonic thymocytes but rather to some maturation process of the thymic lymphocytes starting from 17 days of embryonic life. The influence of extrathymic factors on the turning on of the expression of the 6 S DNA ligase in the post-natal environment is also suggested.

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.

Effects of antileukemic agents on the activity of terminal deoxynucleotidyl transferase from human normal thymic and leukemic cells "in vitro".

Several widely used and experimental antileukemic drugs have been tested on the activity of Terminal deoxynucleotidyl Transferase (TdT) purified from normal human thymocytes and T-derived acute lymphoblastic leukemia peripheral blood lymphoblasts. The majority of these inhibitors were equally potent inhibitors of the enzyme from thymocytes or leukemic lymphocytes. Adriamycine and etoposide were more potent inhibitors of the enzyme purified from thymocytes. Vincristine was a more potent inhibitor of TdT extracted from leukemic lymphocytes than from thymocytes. These results are discussed in terms of possible functions for TdT in the two types of cells and on the value of TdT as an indicator for clinical treatment.