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Biomedical subjects

E Wawra

Publications and source records attributed to E Wawra.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed1998

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.

Biomarkers, Tumor↗
PubMed1996

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.

Animals↗
PubMed1996

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.

Carrier Proteins↗
PubMed1996

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.

Biomarkers↗
PubMed1996

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.

Carrier Proteins↗
PubMed1995

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.

Blotting, Western↗
PubMed1994

Different regulation of thymidine kinase during the cell cycle of normal versus DNA tumor virus-transformed cells.

We compared the cell cycle regulation of thymidine kinase (TK) after centrifugal elutriation in normal human and mouse cells (primary cells, diploid fibroblasts) with its expression in cells transformed with different DNA tumor viruses. Normal cells showed a rise of TK enzyme activity near the G1/S boundary, which peaked in S phase, and in G2 returned approximately to the level of G1. Conversely, in cells derived from viral transformation, TK activity remained high throughout S and G2 phases, although it was induced to a comparable extent at the onset of DNA replication. In addition, transformed cells exhibited much more enzyme activity during all phases of the cell cycle. The observed differences in expression were due neither to different rates of protein turnover nor to differences in enzyme stability. Enzyme activity was always totally paralleled by the protein level. In all normal cells, the pattern of TK mRNA variation during the cell cycle was similar to that of enzyme activity. In all transformed lines, however, mRNA levels were higher and did not fluctuate throughout the cell cycle. Recently we showed (Ogris et al., 1993) that the E2F binding site present in the TK promoter is a target for trans activation of the TK gene by polyoma virus large T antigen. Using cells expressing this antigen under the control of a hormone-inducible promoter, we were able to switch TK cell cycle expression from the normal to the transformed status. Obviously, DNA tumor viruses suppress transcriptional down-regulation of the endogenous DNA precursor pathway enzyme TK during the eukaryotic cell cycle, maybe to improve conditions for their own replication.

Animals↗
PubMed1994

A common regulation of genes encoding enzymes of the deoxynucleotide metabolism is lost after neoplastic transformation.

We determined the cell cycle-dependent fluctuation of mRNAs that encode different enzymes of the deoxynucleotide metabolism in permanent cell lines of human and murine origin. In normal growing cells, dihydrofolate reductase, thymidine kinase, and both subunits of ribonucleotide reductase all show exactly the same variation. The mRNAs rise near the G1-S boundary, peak in early S phase, and return in G2 to approximately the level of early G1. Deoxycytidine kinase mRNA does not follow this pattern, but remains essentially unchanged. Conversely, in DNA tumor virus-transformed cells, the levels of all these mRNAs remain relatively constant throughout all phases. These data provide evidence that DNA tumor viruses suppress a transcriptional down-regulation common to enzymes responsible for the DNA precursor pathway. The usefulness of analysis of mRNA levels of these genes for the detection of DNA tumor virus transformation is indicated.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

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.

Cell Count↗
PubMed1993

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.

Cell Transformation, Viral↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1986

The inhibitory effect of 4-hydroxy-nonenal on DNA-polymerases alpha and beta from rat liver and rapidly dividing Yoshida ascites hepatoma.

The purpose of this study was to determine firstly whether the isolated enzyme DNA polymerase alpha, which functions within the DNA replicase system, exhibits different sensitivity against the thiol-blocking agent 4-hydroxy-nonenal (HNE) when adult rat liver and the rapidly dividing Yoshida ascites hepatoma were used as enzyme sources and, secondly, whether the reaction catalysed by DNA polymerase is the most sensitive step of the DNA replicase system of native cells. DNA polymerase alpha as well as the non-replicative DNA polymerase beta, isolated from both sources, were remarkably similar with regard to their sensitivity against HNE, as indicated by the incorporation of radioactive label from [3H]deoxy-thymidine-triphosphate into DNA. The transport of [14C]thymidine through the plasma membrane and the incorporation of this precursor into DNA were studied with neonatal hepatocytes and with hepatoma cells. The incorporation of thymidine was inhibited at lower concentrations of HNE in both cell lines than the transport process and the reaction catalysed by DNA polymerase alpha. It was concluded that in the DNA replicase system of native liver and hepatoma cells another process different from the reaction catalysed by DNA polymerase alpha is more sensitive to HNE.

Aldehydes↗
PubMed1983

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.

Animals↗