Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism?

Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the G1/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.

Prospective evaluation of postoperative pain in cats undergoing ovariohysterectomy by a midline or flank approach.

Twenty entire female cats were randomly assigned to two groups of 10; the cats in one group underwent ovariohysterectomy by a midline approach and the cats in the other group by a flank approach. Cats were assessed for signs of pain and scores were assigned pre- and postoperatively. There was a tendency for the cats neutered by a flank approach to be in more pain postoperatively (P=0.05). The final pain score for cats in either group was equal to or lower than their baseline score.

The role of p16 in the E2F-dependent thymidine kinase regulation.

The role of alterations of the MTS1 tumor suppressor gene on chromosome 9p21, which encodes p16, the inhibitor of cyclin-dependent-kinase-4 and 6, in tumorigenesis is not yet clear. Phosphorylation of the retinoblastoma protein by cyclin-dependent kinases 4 and 6 prevents its interaction with the transcription factor E2F, which subsequently promotes the expression of S phase regulated genes, such as thymidine kinase. Although a role of p16 in this regulation has been presumed, there is no proof so far that loss of this tumor suppressor gene really affects E2F-mediated regulations. We investigated the regulation of thymidine kinase in phytohemagglutinin-stimulated normal human lymphocytes and in the p16-negative human acute lymphoblastic leukemia cell lines, MOLT-4 and CEM. Compared to normal lymphocytes, MOLT-4 and CEM cells exhibited an altered cell cycle regulation of thymidine kinase, a much higher intracellular activity of this enzyme, and higher thymidine kinase mRNA expression. Transient expression of p16 in normal human lymphocytes caused arrest in G1, but was without effect on the cell growth of MOLT-4 and CEM cells, although all of them express functional retinoblastoma protein. Nevertheless, in the two leukemia cell lines transient overexpression of p16 reestablished the normal regulation of thymidine kinase, paralleled by an increase of the underphosphorylated form of retinoblastoma protein and decrease of free E2F bound to its motif in the thymidine kinase promoter. We demonstrate that loss of p16 causes upregulation of this DNA precursor pathway enzyme via activation of E2F by a mechanism involving retinoblastoma protein.

Cell cycle regulation of deoxycytidine kinase. Evidence for post-transcriptional control.

Deoxycytidine kinase enzyme activity and deoxycytidine kinase mRNA content were determined at different positions of the cell cycle in permanent cell lines. There was no variation of deoxycytidine kinase activity during the cell cycle in two cell lines, whereas in two other lines the enzyme activity was induced (10- and 15-fold) at the G1/S boundary. In contrast, the level of deoxycytidine kinase mRNA never displayed any cell cycle-dependent changes. The decay of enzymatic activity was measured after addition of cycloheximide in different phases of the cell cycle; the enzyme was much more stable in cells with constant activity. We suggest that post-transcriptional mechanisms account for the periodic behaviour of the enzyme activity, and that whether this regulation can be detected depends on the half-life of deoxycytidine kinase.

Specific transformation abolishes cyclin D1 fluctuation throughout the cell cycle.

We analysed cyclin D1 mRNA and protein expression in several different cell types after separating these cells according to their different cell cycle phases by centrifugal elutriation. In normal human and rat fibroblasts cyclin D1 expression is high in early to mid G1 and decreases about 6-7 fold before onset of replication. It has been demonstrated that specific transforming events, such as loss of functional retinoblastoma protein, overexpression of c-myc, and transfection with the human papillomavirus oncoproteins E6 and E7 cause transcriptional downregulation of cyclin D1 expression in logarithmically growing cells. We found that such transformed cells exhibit loss of the cell cycle-dependent cyclin D1 fluctuation accompanied with reduced upregulation of cyclin D1 in G1 phase. The data presented here provide the experimental support for a recently suggested model involving the function of the retinoblastoma protein in cyclin D1 cell cycle regulation.

Expression of the cyclin-dependent kinase inhibitor p16 during the ongoing cell cycle.

It has been demonstrated that protein expression of p16, the inhibitor of cyclin-dependent kinase 4 and 6, increases 4 fold at the G1/S transition when serum-arrested cells are restimulated to logarithmic growth. We examined the cell cycle regulation of this cyclin-dependent kinase inhibitor in cells separated according to their cell cycle phases by centrifugal elutriation. Neither p16 mRNA nor its protein expression are regulated during the cell cycle of normal phytohemagglutinin-stimulated lymphocytes, retinoblastoma protein-negative cells, papilloma virus-transformed cells, and acute promyelocytic leukemia cells. p16 mRNA is constitutively expressed in cells in which we detected the normal E2F-dependent S-phase specific expression of thymidine kinase mRNA. We further observed a G1-phase specific expression of cyclin D1 mRNA in the same cells separated by centrifugal elutriation.

Thymidine kinase is expressed differently in transformed versus normal-cells - a novel test for malignancy.

Using a cytofluorometric assay, we studied the time course of thymidine kinase activity during the cell-cycle in logarithmically growing cells. Two different patterns were found. Many cells, including all strains of normal diploid fibroblasts, exhibited a transient stimulation of enzyme activity during early S-phase. On the contrary, virally transformed cells or lines derived from tumors also increased enzyme activity during G1/S, but kept this level until mitosis. In the latter case, the absolute enzyme activity was significantly higher during all phases of the cell-cycle than in normal cells. Beside its importance for the understanding of the transformed state, this phenomenon has obvious benefits for tumor diagnosis.

Loss of the p16/MTS1 tumor suppressor gene causes E2F-mediated deregulation of essential enzymes of the DNA precursor metabolism.

Homozygous deletions of the tumor suppressor gene p16/MTS1 were reported in a wide variety of tumors and tumor cell lines. Its product inhibits the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and CDK6. Because phosphorylation of pRb is a major regulatory event in the activation of the transcription factor E2F, a role for p16 in the regulation of E2F-dependent transcription was presumed. We investigated the effect of the loss of p16 on E2F-mediated transcription in a tumor progression model consisting of three cell lines originating from a common precursor cell--one p16-positive cell line established from the primary biopsy and two lines derived from more advanced stages of the tumor representing the same cell clone after loss of p16. We observed up- and deregulation of E2F-dependent transcription during the cell cycle of the p16-negative cell clones, which returned to normal after transient expression of p16. This p16-dependent regulation affects a set of enzymes necessary for the activation of all four DNA precursors; it is paralleled by the interconversion of transcriptionally active free E2F and transcriptionally inactive higher molecular complexes of E2F and is dependent on the existence of endogenous pRb. Furthermore, we show that p16-negative cell clones exhibit a growth advantage compared to their p16-positive counterparts. One might speculate that one feature of tumor progression could be deregulation of E2F-dependent transcription caused by loss of p16.

Substratwahlversuche mit Microhedyle milaschewitchii Kowalevsky (Gastropoda, Opisthobranchia: Acochlidiacea).

Microhedyle milaschewitchii Kow. obtained from a sublittoral shellsand near Rovinj (Yugoslavia) prefers a grain size range of 0.5 to 2 mm in simple-choice experiments. This fraction predominates in the natural substrate (73.1%). Light did not affect the behaviour of the animals which are much less thigmotactical than Protodrilus. Sterile sands are rejected when offered together with natural substrate, but are equally attractive after having been incubated with sand bacteria for ten days. Similarly dried sand is not accepted by the animals. There is no significant preference between quartz and chalk sands, both containing bacteria. Optimal grain size and living microorganisms apparently are essential for the colonization of a substrate.

Thymidine kinase expression. A marker for malignant cells.

The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to discriminate between normal growing cells and virally transformed cells or lines derived from tumours. In material (blood and bone marrow) taken from leukaemia patients, we identified the leukaemic cells in a surplus of normal leucocytes. From cell cultures representing a tumour progression model, only the later, and malignant, stages showed enhanced fluorescence, whereas benign tumour cells looked normal.

Microinjected deoxynucleotides for the study of chemical inhibition of DNA synthesis.

Microinjection is shown to be a useful tool for studies of chemical inhibition of DNA synthesis: inhibitor-treated cells were injected with combinations of radioactive precursors and their uptake into DNA was monitored by autoradiography. The results obtained from inhibition by cytosinearabinoside, aphidicolin, trifluorothymidine, and fluorodeoxyuridine agreed well with the common knowledge about these drugs. Short-term (but not long-term) treatments with methotrexate were compensated by injections of thymidine-nucleotides. The effect of hydroxyurea was in part, but not fully, reversed by injection of all four deoxytriphosphates; this implies a second mechanism besides inhibition of ribonucleotide reductase. Regulation of reductase was responsible for the effect of thymidine: the enhanced dTTP caused a depletion of dCTP and dATP. Novobiocin was different from all other drugs tested, DNA polymerase or enzymes of the precursor metabolism are obviously not targets of this drug.

Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled.

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.

Evidences for the function of DNA polymerase-beta in unscheduled DNA synthesis.

The activities of DNA polymerase-alpha and -beta isolated from pig spleen were determined at different temperatures and in the presence of different concentrations of inhibitors. The results were compared with parallel estimations of replicative DNA synthesis and UV-induced repair synthesis in spleen cells. In respect to pCMB and aCTP, polymerase-alpha is more sensitive than polymerase-beta and similarly is replication more sensitive than repair. Repair synthesis and the activity of polymerase-beta decreases at temperatures higher than 40 degrees C whereas both replication and the activity of polymerase-alpha are greatly stimulated at elevated temperatures with optima of 45 degrees C (polymerase-alpha) and 41 degrees C (replication). The results favour the hypothesis that polymerase-beta is involved in repair synthesis.

Cytofluorometric assay for the determination of thymidine uptake and phosphorylation in living cells.

Thymidine kinase is a key enzyme for the application of drugs in chemotherapy and for diagnosis. Although of great interest, its regulation during cell cycle and differentiation is difficult to study, as current techniques for isolation of cells in different phases of growth are unsatisfactory. An assay that allows the determination of enzymatic activity in situ in single cells would be much faster than present methods and would elegantly avoid synchronization procedures. We synthesized different analogues of thymidine with the 5-methyl group substituted by a fluorochrome. At least three of these compounds were phosphorylated by thymidine kinase in cell free extracts and were taken up and phosphorylated by cells in culture. The cytofluorometric signal of the accumulated fluorochrome in any given cell reflected the thymidine kinase activity of this cell. Simultaneous measurement of cell-cycle dependent parameters allowed the correlation of thymidine kinase activity with the phase of growth in mixed cell populations.

Cytofluorometric determination of thymidine kinase activity in a mixture of normal and neoplastic cells.

A cytofluorometric assay allowing the measurement of thymidine phosphorylation in single cells had been established (Hengstschläger & Wawra, 1993). This assay enables us to correlate intracellular thymidine kinase (TK) activity with the DNA content of single cells. Enzyme activity levels from neuroblastoma cells and normal fibroblasts derived from the same patient were determined. Using this cytofluorometric assay in a mixture of both cell types the neoplastic cells could be distinguished from the normal fibroblasts because of their higher TK level. A human lymphoblastoid cell line was compared with the cell line KG-1, derived from an acute myelogenous leukaemia, in the same way. The increased enzyme activity enabled us to detect KG-1 cells in a mixture with an 10,000-fold excess of Epstein Barr virus transformed lymphocytes.

[The action of hyperthermia on DNA repair (author's transl)]. / Die Wirkung von Hyperthermie auf DNA-Reparaturvorgänge.

The time-course of DNA repair after gamma irradiation was measured in HeLa cells at various temperatures. Unscheduled DNA synthesis was estimated by incorporation of 3H-thymidine in presence of hydroxyurea. To detect the ligase reaction, the number of single strand breaks (SSB) was determined by centrifugation in alcaline sucrose as well as by hydroxylapatite chromatography after partial denaturation. In addition, the temperature dependence of DNA polymerase and DNase reaction in cell-free systems were measured. These data were compared with the reduction of colony-forming ability of the cells caused by gamma irradiation and following repair at various temperatures. All steps of repair proceed faster at 41--43 degrees than at 37 degrees but cells are most resistant to gamma irradiation at 37 degrees. We therefore assume that the DNA repair process at 42 degrees is faster but more error prone than at 37 degrees.

Identification of leukaemic cells in bone marrow and blood samples by a new cytofluorometric assay.

The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. We used this fact to detect neoplastic cells in samples freshly taken from leukaemia patients and kept frozen in liquid nitrogen until analysis. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to identify leukaemic cells in a surplus of normal ones. Our results demonstrate the benefits of this assay for leukaemia diagnosis.

Chicken histone H5 inhibits transcription and replication when introduced into proliferating cells by microinjection.

Chicken erythrocyte histone H5 has been suggested repeatedly to be a general suppressor of transcription and replication. Therefore, the biological functions of H5 were investigated and compared with those of H1 (H1a + H1b) by microinjection of the purified proteins into proliferating L6 rat myoblasts. By pulse-labelling of the injected cells with [3H]uridine and [3H]thymidine it was shown that H5 blocked both transcription and replication substantially, and that the chromatin of the injected cells became densely compacted. H1 also suppressed these functions, but to a much lesser degree. The effects were specific and not caused by change in intracellular pH caused by introduction of the very basic H5, or its non-specific interaction with nucleic acid, since injection of protamine or lysozyme did not affect the cells. The migration and localization of injected H5 was monitored at different times after injection by immunofluorescence, which revealed that H5 was efficiently and stably concentrated in the nucleus. The results indicate that H5 indeed might function as an inactivator of the erythroid genome in its natural environment, probably by keeping the chromatin in a very condensed state.

[Passive smoking at the workplace--injurious to health? (author's transl)]. / Das Passivrauchen am Arbeitsplatz--eine Gesundheitsschädigung?

The question as to whether passive smoking at the workplace is injurious to health is dealt with. With the aid of the bibliographic standard literature on industrial, social and preventive medicine, all experimental investigations of this subject and published since 1970 were considered. The important results and findings are presented in tabulated surveys broken down by the substances contained in smoke. The present results of investigation are discussed with respect to a practical workplace model whose air-hygienic conditions are determined by legal prescriptions for workplaces. The measured concentrations of the particulate mass and of the most important individual constituents of smoke, carbon monoxide, nicotine and aldehyde make it highly unlikely that passive smoking at the workplace causes injury to health if the prevailing regulations governing the workplace are complied with. Particular attention has to be given to the occupational problems existing in night bars, restaurants and discothecs.