Induction of high polyploidy in Phaseolus cell cultures by the protein kinase inhibitor,K-252a

Summary Somatic polyploidy of species-specific and tissue-specific degrees occurs in almost all plant species studied so far, but nearly nothing is known about the control mechanisms switching the mitotic cycle to an endoreduplication cycle. In order to search for a possible role of the cdc2 kinase, cell suspension cultures of the Runner bean, Phaseolus coccineus (Leguminosae) were treated with K-252a, an inhibitor of protein kinase activity. The treatment resulted in continuous cell cycles without mitosis, and hence induced polyploidy levels up to 2048C. It is, therefore, suggested that phosphorylation of a protein kinase, probably of the cell cycle-important p34^cdc2 type, is involved in the control of endoreduplication.     

Induction of thymidine kinase in enzyme-deficient chinese hamster cells

Previous work with Chinese hamster cells suggests that thymidine kinase deficiency and loss of potential for plating in HAT medium may arise by a process of mutation coupled with site-specific repression by bromodeoxyuridine at the tk locus. In this study, tk^? Chinese hamster cells were exposed to a series of inductors to determine whether revertants for the putative second stage originate by genetic or epigenetic change. Brief exposure to 5-azacytidine resulted in massive conversion to the HAT⁺ state, and revertants showed levels of thymidine kinase activity intermediate between those of tk^? and wild-type cells. By contrast, incidence of HAT⁺ cells rose only slightly in populations mutagenized with ethyl methanesulfonate. Large increases in frequency of HAT⁺ cells were obtained by treatment with n-butyrate and L-ethionine, which affect gene expression in other cell systems but have no known mutagenic potential. Induction of HAT⁺ revertants seems to be mediated by a stable epigenetic shift, which reverses the gradual extinction of thymidine kinase activity in the parent cells. The data support the view that induction in Chinese hamster cells results from changes in DNA methylation patterns, and suggest studies to define the process in molecular terms.     

Treatment of glioblastoma by direct inoculation of concentrated high titer-recombinant retrovirus carrying the herpes simplex virus thymidine kinase gene.

We prepared retroviruses carrying the lacZ gene or herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4-2.5 x 10(11) colony-forming units (cfu)/ml, and stereotaxically inoculated only 3 microliters of the retroviruses into a mouse glioma model. This resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty per cent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas. To achieve further safety in the gene therapy of glioma, genes abundantly expressed in human glioblastoma were searched by the Serial Analysis of Gene Expression (SAGE) technique. Among the top-147 most expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, originated from an unidentified gene, which was not detected in human astrocyte cultures. Real-time quantitative RT-PCR showed that MAGE-E1 expression was 2.6-15 fold enriched in glioblastoma relative to human astrocytes. Expressed Sequence Tags (ESTs) containing this tag were homologous to melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by 5'-rapid amplification of cDNA ends (RACE) technique. MAGE-E1 expression was enriched in glioblastoma and low in other cancers, and MAGE-E1 expression was detected only in brain and ovary among normal tissues. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family, which can be applied to glioma-specific gene transduction.     

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Evolution by polyploidy and gene regulation in Anura

The evolution of the metazoa has been characterized by gene redundancy, generated by polyploidy, tandem duplication and retrotransposition. Polyploidy can be detected by looking for duplicated chromosomes or segments of orthologous chromosomes in post-polyploid animals. It has been proposed that the evolutionary role of polyploidy is to provide extra-copies of genes, whose subsequent alteration leads to new functions, increased biological complexity, and, ultimately, speciation. We review the theory of evolution by genome duplication, basing our arguments on findings from autopolyploid anurans and fish, undergoing post-polyploidy diploidization. We conclude that: 1) the high genetic variability of autotetraploid anurans is a result of tetrasomic expression, based on studies of isozymes and other proteins. 2) Epigenetic mechanisms mediate the reduced expression or silencing of redundant copies of genes in the regulation of gene expression of these tetraploids. This conclusion is based on data concerning ribosomal and hemoglobin gene activity. 3) Duplication of the genome may have occurred more than once in the phylogeny of the anurans, as exemplified by 4n and 8n Leptodactylidae species.     

Somatic polyploidy examined by flow cytometry in Daphnia

Flow cytometry was used to examine the patterns of somatic polyploidyand to determine the haploid genome sizes in Daphnia. The averageproportions of polyploid nuclei among adult animals of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 27,24, 24 and 24%, respectively. Both interclonal and developmentallyregulated variation were observed in the level of somatic polyploidy.Adult animals expressed more extensive polyploidy than did juveniles.The estimates of the haploid genome sizes (C values) of D.pulex,D.longispina, D.pulex x D.longispina and D.magna equalled 0.32,0.27, 0.26 and 0.37 pg, respectively. These are the smallestgenomes reported in crustaceans. Apparently, somatic polyploidycompensates for the loss of genetic material and gives riseto genetic plasticity which may contribute to the considerablelevel of phenotypic plasticity observed in Daphina.     

Megakaryocyte polyploidy is inhibited by lysyl oxidase propeptide

Megakaryocytes (MKs), the platelet precursors, undergo an endomitotic cell cycle that leads to polyploidy. Lysyl oxidase propeptide (LOX-PP) is generated from lysyl oxidase (LOX) pro-enzyme after proteolytical cleavage. We recently reported that LOX, a known matrix cross-linking enzyme, contributes to MK lineage expansion. In addition, LOX expression levels are ploidy-dependent, with polyploidy MKs having minimal levels. This led us to test the effects of LOX-PP on the number and ploidy of primary MKs. LOX-PP significantly decreases mouse bone marrow MK ploidy coupled with a reduction in MK size. MK number is unchanged upon LOX-PP treatment. Analysis of LOX-PP- or vehicle-treated MKs by western blotting revealed a reduction in ERK1/2 phosphorylation and in the levels of its downstream targets, cyclin D3 and cyclin E, which are known to play a central role in MK endomitosis. Pull-down assays and immunochemistry staining indicated that LOX-PP interacts with α-tubulin and the mictotubules, which can contribute to decreased MK ploidy. Thus, our findings defined a role for LOX-PP in reducing MK ploidy. This suggests that high-level expression of LOX in aberrantly proliferating MKs could play a part in inhibiting their polyploidization via LOX-PP.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Transition state analysis of the arsenolytic depyrimidination of thymidine by human thymidine phosphorylase

Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, promotes angiogenesis, and is involved in metabolic inactivation of antiproliferative agents that inhibit thymidylate synthase. Understanding its transition state structure is on the path to design transition state analogues. Arsenolysis of dT by hTP permits kinetic isotope effect (KIE) analysis of the reaction by forming thymine and the chemically unstable 2-deoxyribose 1-arsenate. The transition state for the arsenolytic reaction was characterized using multiple KIEs and computational analysis. Transition state analysis revealed a concerted bimolecular (A(N)D(N)) mechanism. A transition state constrained to match the intrinsic KIE values was found using density functional theory (B3LYP/6-31G*). An active site histidine is implicated as the catalytic base responsible for activation of the arsenate nucleophile and stabilization of the thymine leaving group during the isotopically sensitive step. At the transition state, the deoxyribose ring exhibits significant oxocarbenium ion character with bond breaking (r(C-N) = 2.45 ?) nearly complete and minimal bond making to the attacking nucleophile (r(C-O) = 2.95 ?). The transition state model predicts a deoxyribose conformation with a 2'-endo ring geometry. Transition state structure for the slow hydrolytic reaction of hTP involves a stepwise mechanism [Schwartz, P. A., Vetticatt, M. J., and Schramm, V. L. (2010) J. Am. Chem. Soc. 132, 13425-13433], in contrast to the concerted mechanism described here for arsenolysis.     

Effects of polyploidy on photosynthesis

In polyploid plants the photosynthetic rate per cell is correlated with the amount of DNA per cell. The photosynthetic rate per unit leaf area is the product of the rate per cell times the number of photosynthetic cells per unit area. Therefore, the photosynthetic rate per unit leaf area will increase if there is a less than proportional increase in cell volume at higher ploidal levels, or if cell packing is altered to allow more cells per unit leaf area. In autopolyploids (Medicago sativa, C₃ species, and Pennisetum americanum, C₄ species) there is a doubling of photosynthesis per cell and of cell volume in the tetraploid compared to the diploid. However, there is a proportional decrease in number of cells per unit leaf area with this increase in ploidy such that the rate of photosynthesis per leaf area does not change. There is more diversity in the relationship between ploidal level (gene dosage) and photosynthetic rates per unit leaf area in allopolyploids. This is likely to reflect the effects of natural selection on leaf anatomy, and novel genetic interactions from contributed genomes which can occur with allopolyploidy. In allopolyploid wheat (C₃ species) a higher cell volume per unit DNA at the higher ploidal level is negatively correlated with photosynthesis rate per unit leaf area. Although photosynthesis per cell increases with ploidy, photosynthesis per leaf area decreases, being lowest in the allohexaploid, cultivated bread wheat (Triticum aestivum). Alternatively, doubling of photosynthetic rate per cell with doubling of DNA, with apparent natural selection for decreased cell volume per unit DNA, results in higher rates of photosynthesis per leaf area in octaploid compared to tetraploid Panicum virgatum (C₄) which may be a case of allopolyploidy. Similar responses probably occur in Festuca arundinacea. Therefore, in some systems anatomical factors affecting photosynthesis are also affected by ploidal level. It is important to evaluate that component as well as determining the effect on biochemical processes. Current information on polyploidy and photosynthesis in several species is discussed with respect to anatomy, biochemistry and bases for expressing photosynthetic rates.Abbreviations Chl chlorophyll - RuBPC ribulose-1,5-bisphosphate carboxylase     

Induction of cell death in activated hepatic stellate cells by targeted gene expression of the thymidine kinase/ganciclovir system

Liver fibrosis is the result from a relative imbalance between synthesis and degradation of matrix proteins. Following liver injury of any etiology, hepatic stellate cells undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Upon this cellular transdifferentiation the effector cell becomes the major source of fibrillar and non-fibrillar matrix proteins resulting in excessive scar formation and cirrhosis, the end stage of fibrosis. Concomitant with progressive liver fibrosis, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is strongly activated in hepatic stellate cells. We have developed a recombinant replication-defective adenovirus in which the TIMP-1 promoter is coupled to the herpes simplex virus thymidine kinase gene rendering activated hepatic stellate cells susceptible to ganciclovir. This novel targeted suicide gene approach was validated in a culture model considered to reflect an accelerated time course of the cellular and molecular events that occur during liver fibrosis. We demonstrate that transfer of the suicide gene to culture-activated hepatic stellate cells results in a strong expression of the respective transgene as assessed by Northern blot and Western blot analyses. The enzyme catalyzed the proper conversion of its prodrug subsequently initiating programmed cell death as estimated by caspase-3 assay and Annexin V-Fluos staining. Altogether, these results indicate that induction of programmed cell death is a promising approach to eliminate fibrogenic HSC.     

Induction of thymidine kinase synthesis by 5-androstene-3 beta,17 beta-diol in the uterus of the immature rat

In uteri from immature female rats, thymidine kinase activity was largely increased by administration of 5-androstene-3 beta, 17 beta-diol. Kinetic studies showed that the enzyme activity reached its maximum level 30 h after hormone administration. That increase in thymidine kinase activity was dose-dependent and could be related to the synthesis of new molecules of enzyme. Moreover, it was exclusively observed in target-organs for estrogens. It was concluded that 5-androstene-3 beta, 17 beta-diol which results from the metabolism of dehydroepiandrosterone had estrogen-like properties with regard to the induction of thymidine kinase.     

The evolutionary consequences of polyploidy

Polyploidization, the addition of a complete set of chromosomes to the genome, represents one of the most dramatic mutations known to occur. Nevertheless, polyploidy is well tolerated in many groups of eukaryotes. Indeed, the majority of flowering plants and vertebrates have descended from polyploid ancestors. This Review examines the short-term effects of polyploidization on cell size, body size, genomic stability, and gene expression and the long-term effects on rates of evolution.