DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Analysis of 5'-ends of short DNA fragments excreted by phytohemagglutinin stimulated lymphocytes

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and the excreted DNA was isolated from the medium after four days of incubation of cells. The excreted DNA was labeled at the 5'-end with [gamma-32P]ATP and polynucleotide kinase. Analysis of the end-labeled material revealed a size distribution with a chain length of 6 - 60 nucleotides. These short DNA fragments did not contain ribo-nucleotides at their 5'-termini. P1 nuclease digestion did not release specific deoxyribonucleoside monophosphates from the 5'-end of the excreted DNA fragments. These results point to the non-specific degradation of DNA excreted by stimulated lymphocytes.     

Cytidine 5''-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes.

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h.     

Protein synthesis in resting and stimulated human lymphocytes

Summary The ribosomal profiles in lysates from resting and phytohemagglutinin stimulated human lymphocytes have been analyzed by sucrose gradient centrifugation. The percentage of polyribosomes increased during lymphocyte transformation reaching a maximal value of 60 to 70% of the total ribosomes after 72 hours of mitogen addition. This time period coincides with maximalin vivo protein synthesis. On the other hand, in nonstimulated lymphocytes, about 25% of the ribosomal particles appeared as aggregates, independently of the incubation period.Experiments performed with homologous cell free systems containing ribosomes and supernatant fluids prepared from unstimulated or activated lymphocytes demonstrate that the mixtures containing both components from stimulated lymphocytes are several fold more active in polypeptide synthesis than the systems which contain ribosomal particles and cell sap from resting cells. Assays carried out with mixtures combining the components from both sources indicate that the increased activity depends on ribosomes as well as on the supernatant fractions.Dedicated to Professor LUIS F. LELOIR on the occasion of his 70^th birthday.     

Cell kinetics and radiosensitivity of Cercopithecus aethiops sabaceus lymphocytes stimulated by phytohemagglutinin

The use of the BudR-Giemsa technique has shown that the peripheral blood lymphocytes of Cercopithecus aethiops sabaceus display a very fast answer to PHA-stimulation. At 32 h culture time numerous mitoses are already present and at 48 h more than 30% of the dividing cells are in second mitosis. Exposure to X-irradiation results in a higher incidence of unstable aberrations than in human lymphocytes.     

Lymphotoxin production by human lymphocytes. I. Study of the supernatants of phytohemagglutinin stimulated human lymphocyte cultures]

Lymphotoxin was found to be present in supernatants from 22 human lymphocytes cultures stimulated with phytohemagglutinin in a dose of 5 and 10 microgram/ml. The lymphocytes were obtained from the peripheral blood of 6 apparently healthy persons. Lymphotoxin activity was determined by simple and objective method, i.e. by staining the target cells (mouse L-cells) monolayer with crystal violet, with the following determination of optic densities of the L-cells lysates at 570 nm in the spectrophotometer. As revealed, 1 : 5 dilutions of the supernatants from the lymphocyte cultures incubated for 48 hours inhibited the L-cells growth by from 40 to 60%. With further incubation of the cultures (up to 72 and 96 hours) the cytotoxicity of their supernatants for the target cells showed no increase, whereas the blasttransformation index reaches the maximal value by 72nd incubation hour. Supernatants from unstimulated lymphocyte cultures failed to produce any cytotoxic effect on L-cells.     

DNA synthesis and proliferation of human lymphocytes in vitro. II. Characterization of the DNA newly synthesized after phytohemagglutinin stimulation

DNA newly synthesized by phytohemagglutinin-(PHA) stimulated human lymphocytes has been analyzed for the possibility that all DNA synthesis may not represent premitotic genome duplication. Equilibrium density gradient characterization of bromodeoxyuridine-(BUdR) substituted DNA demonstrates semi-conservative DNA replication, without evidence for repair synthesis. Experiments to detect selective replication (amplification) of a portion of the DNA involved measurement of reassociation kinetics as well as measurement of the kinetics of BUdR appearance in prelabeled DNA. The presence of large quantities of amplified DNA has been excluded, although amplification cannot be ruled out completely by these techniques. Finally, the kinetics of DNA release from dead lymphocytes is characterized, and factors tending to reduce the complexity of released, labeled DNA are identified.     

Early effects of phytohemagglutinin on induction of DNA polymerase, thymidine kinase, deoxyribonucleoside triphosphate pools and DNA synthesis in human lymphocytes.

In phytohemagglutinin stimulated human lymphocytes the time relationship was determined between induction of the parameters mentioned. The results indicate that the induction occurred in a specific sequence. Thus, a simultaneous increase in the activity of DNA polymerase and thymidinekinase occurred after 15 h of incubation with Phytohemagglutinin. Furthermore, this enhancement occurred 2 h before the expansion of the TTP and dCTP pools and 4 h before the expansion of the dATP and dGTP pools. The rate of [3H] deoxyguanosine incorporation into DNA increased simultaneously with the expansion of the TTP and dCTP pools.     

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes in vitro

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes were biphasic: The chemicals at very low concentrations enhanced DNA synthesis, whereas higher concentrations inhibited DNA synthesis. The concentrations of arsenicals at which the maximum stimulating effect was found were 1×10^?5 M, 1×10^?6 or 2×10^?6 M, and 0.8×10^?6 or 1×10^?6 M for sodium arsenite exposure of 1 h, 3 d, and 6 d, respectively; for sodium arsenate, 1× 10^?5 M, 1×10^?5 M, and 2×10^?6 or 5×10^?6 M, respectively. Arsenicals must be present for the entire 6-d culture period to produce maximum stimulation of DNA synthesis in human lymphocytes. The longer exposure of the lymphocytes to arsenicals, the lower the concentrations of arsenicals at which the maximum stimulating effect on DNA synthesis was found. Stimulating effect of trivalent arsenic (sodium arsenite) on DNA synthesis was stronger than pentavalent arsenic (sodium arsenate), and the stronger the effect of trivalent arsenic than pentavalent, the longer exposure of the cells to the chemicals. Both sodium arsenite and sodium arsenate stimulated DNA synthesis in human lymphocytes to a lower degree than phytohemagglutinin (PHA).