Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

花生种子萌发早期胚轴细胞DNA合成的特点

花生种子吸胀6h后胚轴DNA中有~3H-胸苷掺入。咖啡因和羟基脲均对6～12h的~3H—胸苷掺入具强烈的抑制作用;当12～24h时,咖啡因的抑制作用较大;但30h以后,羟基脲的抑制作用超过咖啡因。双链DNA放射性从种子吸胀9h后迅速上升,单链DNA放射性在吸胀12h后出现一个明显的峰。但在吸胀12h后,单链DNA形成和存在的时间是短暂的。     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5:10(-5) per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10(-6) in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with gamma-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than gamma-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

Parameters of unbalanced growth and reversible inhibition of deoxyribonucleic acid synthesis in Brevibacterium ammoniagenes ATCC 6872 induced by depletion of Mn2+. Inhibitor studies on the reversibility of deoxyribonucleic acid synthesis

Unbalanced growth induced by depletion of manganese ions was a prerequisite for production of ribonucleotides in a high salt mineral medium with the wildtype strain Brevibacterium ammoniagenes ATCC 6872. The concentration of manganese strictly controlled the overall deoxyribonucleic acid (DNA) synthesis, whereas ribonucleic acid (RNA), protein and cell wall synthesis remained essentially unimpaired in the manganese-lacking cells.The reversibility of inhibition of overall DNA synthesis was shown by enhanced incorporation (up to threefold compared to the cultures supplied with sufficient manganese) of [8-¹⁴C] adenine into alkali-stable, trichloroacetic acid-insoluble material after subsequent addition of 10 M MnCl₂ to 15 h-old depleted cultures.The results of inhibitor studies on the restoration of overall DNA synthesis due to subsequent addition of manganese ions to depleted cultures suggest that ribonucleotide reduction is the primary target of the manganese starvation during nucleotide fermentation with Brevibacterium ammoniagenes ATCC 6872.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

Algal oxidation of aromatic hydrocarbons: formation of 1-naphthol from naphthalene by Agmenellum quadruplicatum, strain PR-6.

Hydroxyurea induces repair replication in human lymphoblastoid NC37 BaEV cells during incubation with liver microsomes and NADPH. Catalase reduces hydroxyurea-induced repair by more than 95%, suggesting that hydrogen peroxide derived from hydroxyurea in the presence of the metabolic activation system is involved in the DNA damage.