Lubos Cipak

Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells.

Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in‐vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in‐vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC100 was below 100 μg mL−1 for HeLa cells and approached a 10 μ mL−1 limit for the leukemia L1210 cells. For both cell lines the IC50 was found to be less than 4 μg mL−1, a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10–50 μ mL−1 berberine, G0/G1 cell cycle arrest was observed. Futhermore, a concentration‐dependent decrease of cells in S phase and increase in G2/M phase was detected. In addition, apoptosis detected as sub‐G0 cell population in cell cycle measurement was proved in 25–100 μg mL−1 berberine‐treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis.

Effects of flavonoids on cisplatin-induced apoptosis of HL-60 and L1210 leukemia cells

Effects of three flavonoids, quercetin (QU), galangin (GA), and chrysin (ChR) on cisplatin (cis-Pt)-induced apoptosis of human promyelocytic leukemia HL-60 cells and murine leukemia L1210 cells were investigated. The quantitative analysis of apoptotic DNA fragmentation was used to show that preincubation of cells with flavonoids can influence cis-Pt-induced apoptosis in different way. ChR had no effect, QU enhanced, and GA reduced apoptotic DNA fragmentation. It is also shown that combined treatment with QU and cis-Pt showed synergistic effect, however, GA combined with cis-Pt exhibited antagonism on cytotoxicity in L1210 murine leukemia cells. We assume that tested flavonoids affect the important biological activities connected with cancer chemotherapy and chemoprevention as they differently modulated the sensitivity of cells to cis-Pt treatment. QU is presented as pro-apoptotic agent and GA as agent with anti-apoptotic potential.

Casein kinase 1 is required for efficient removal of Rec8 during meiosis I

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I. Our data are consistent with the model that Hhp1/Hhp2-dependent phosphorylation of Rec8 is required for separase-mediated cleavage of Rec8 during meiosis I.

Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.

Dual activity of triterpenoids: apoptotic versus antidifferentiation effects

Triterpenoids are natural, biologically active compounds extracted from many plants. They possess antiinflammatory, anticancer, and antioxidant properties. In the report presented, antiproliferative effects and leukemia cell growth and apoptosis modulating activities of ursolic acid (UA) and oleanolic acid (OA) were investigated. Both triterpenoids are inhibitors of leukemia cell growth and inductors of apoptosis. However, when applied in combination with anthracycline antitumor antibiotic doxorubicin (Dox), UA and OA diversely modulate therapeutic efficacy of Dox, due to different antioxidant activities. Compare to OA showing synergism/additive effect with Dox, UA (stronger antioxidant) acts antagonistically and reduces leukemia cell growth inhibiting and differentiation effects induced by Dox. In conclusion, these findings suggest that although triterpenoids UA and OA can induce apoptosis, their antioxidant activities can interfere with the therapeutic effect of antitumor antibiotic Dox which mechanism of action is attributed to the production of reactive oxygen species.

Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe

Reversible protein phosphorylation is a major regulatory mechanism in a cell. A chemical-genetic strategy to conditionally inactivate protein kinases has been developed recently. Mutating a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors. The inhibitor can only bind to the mutant kinase and not to any other wild-type kinase, allowing specific inactivation of the modified kinase. Here, we describe a protocol to construct conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe. This protocol can be completed in about 3 weeks and should be applicable to other organisms as well.

Generation of a set of conditional analog-sensitive alleles of essential protein kinases in the fission yeast Schizosaccharomyces pombe.

The genome of the fission yeast Schizosaccharomyces pombe encodes for 17 protein kinases that are essential for viability. Studies of the essential kinases often require the use of mutant strains carrying conditional alleles. To inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. The mutation of a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. Using this approach, we constructed conditional analog-sensitive alleles of 13 essential protein kinases in the fission yeast S. pombe.

Laser microsurgery provides evidence for merotelic kinetochore attachments in fission yeast cells lacking Pcs1 or Clr4

In order to segregate chromosomes properly, the cell must prevent merotelic kinetochore attachment, an error that occurs when a single kinetochore is attached to microtubules emanating from both spindle poles. Merotelic kinetochore orientation represents a major mechanism of aneuploidy in mitotic mammalian cells and it is the primary mechanism of chromosome instability in cancer cells. Fission yeast mutants defective in putative microtubule-site clamp Pcs1/Mde4 or Clr4/Swi6-dependent centromeric heterochromatin display high frequencies of lagging chromosomes during anaphase. Here, we developed an assay based on laser microsurgery to show that the stretched morphology of lagging kinetochores in pcs1Δ and clr4Δ mutant cells is due to merotelic attachment. We further show that Mde4 is regulated by Cdc2 and that Cdc2 activity prevents precocious localization of Mde4 to the metaphase spindle. Finally, we show that Pcs1/Mde4 complex shares similar features with the conserved kinetochore complex Spc24/Spc25 suggesting that these two complexes may occupy a similar functional niche.

S. pombe genome deletion project: An update

The fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a PCR based strategy. However, deletion of some genes has not been possible using this methodology. Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. The collection of deletion mutants now covers 99% of the fission yeast open reading frames.

Apoptosis induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human leukemia cells involves ROS-mitochondrial mediated death signaling and activation of p38 MAPK.

Benzothiazoles are multitarget agents with broad spectrum of biological activity. 2-Acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile (AMBAN) is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects towards tumor cells. The aim of the present study was to examine the antiproliferative and apoptosis inducing activities of AMBAN towards human leukemia HL60 and U937 cells. Further, the molecular mechanism involved in AMBAN-induced apoptosis was investigated. Benzothiazole inhibited the growth and induced programmed cell death of HL60 and U937 cells. In addition, AMBAN elevated the level of reactive oxygen species, decreased the mitochondrial membrane potential, activated caspases 9 and 3, induced the cytochrome c release and PARP cleavage and led to intranucleosomal DNA fragmentation. Further, p38 MAPK was associated with the apoptotic activity of AMBAN. It can be concluded that AMBAN-induced apoptosis in HL60 and U937 cells through mitochondrial/caspase 9/caspase 3-dependent pathway.