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L Meijer

Publications and source records attributed to L Meijer.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

Antineoplastic Agents, Phytogenic↗
PubMed2006

7-Bromoindirubin-3'-oxime induces caspase-independent cell death.

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser-microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion-specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion-specific expression or regulation.

Animals↗
PubMed2001

Recruitment of cdc2 kinase by DNA topoisomerase II is coupled to chromatin remodeling.

Although initiation of chromosome condensation during early prophase is linked temporally to the appearance of the mitotic cdc2 kinase in the nucleus, it is not known what targets the kinase to the nucleus and how this is coupled to chromatin remodeling. We now report that cdc2 kinase forms stable molecular complexes with the nuclear enzyme DNA topoisomerase II, which is associated with marked stimulation of both DNA binding and catalytic activity of topoisomerase II, albeit in a phosphorylation-independent manner. The molecular interaction is required for recruitment of cdc2 kinase, as shown by incubation of purified enzymes with chicken erythrocyte nuclei, which have neither endogenous topoisomerase II nor cdc2 kinase. The physical association between the two enzymes alters the DNA/topoisomerase II interaction as shown by pulse-field electrophoresis after incubation of intact nuclei with the specific topoisomerase II inhibitor VM-26. Furthermore, the presence of both enzymes, but not either enzyme alone, is accompanied by extensive chromatin remodeling converting the interphase nuclei into precondensation chromosomes with striking resemblance to early prophase structures. Our results reveal a novel property of cyclin-dependent kinases and demonstrate that the recruitment of cdc2 kinase by topoisomerase II is coupled to chromatin remodeling.

Active Transport, Cell Nucleus↗
PubMed2001

Structure-activity relationships and inhibitory effects of various purine derivatives on the in vitro growth of Plasmodium falciparum.

The development of novel chemotherapeutic agents has become an urgent task due to the development and rapid spread of drug resistance in Plasmodium falciparum, the protozoan parasite responsible for cerebral malaria. Cyclin-dependent kinases (CDKs) are essential for the regulation of the eukaryotic cell cycle, and several enzymes of this family have been identified in P. falciparum. In recent years, a number of purine-derived kinase inhibitors have been synthesised, some of which display selective activity against CDKs. This report describes a study in which various purine derivatives were screened for in vitro antimalarial activity. The erythrocytic asexual stages of the chloroquine-resistant P. falciparum strain (FCR-3) were cultivated in vitro in the presence of the various purines, and their effect on parasite proliferation was determined by the [3H]hypoxanthine incorporation assay. Our results show considerable variation in the sensitivity of P. falciparum to the different purines, as well as a general independence from their effect on purified starfish CDK1/cyclin B activity, which has been the standard assay used to identify CDK-specific inhibitors. Two subfamilies of purines with moderate to poor activity against CDK1/cyclin B activity showed submicromolar activity against P. falciparum. Structure-activity analysis indicates that certain structural features are associated with increased activity against P. falciparum. These features can be exploited to synthesise compounds with higher activity and specificity towards P. falciparum.

Adenine↗
PubMed2001

Anti-mitotic properties of indirubin-3'-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest.

The bis-indole indirubin is the active ingredient of the Traditional Chinese Medicine recipe Danggui Longhui Wan used against chronic myelocytic leukemia. We have previously shown that indirubins are potent inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3. We here investigated the anti-mitotic properties of this class of compounds using the cell permeable indirubin-3'-monoxime and the HBL-100 cell line. Indirubin-3'-monoxime reversibly arrests asynchronous HBL-100 cells in G2. This arrest is not accompanied by any significant change in expression of the major cell cycle regulators. However indirubin-3'-monoxime inhibits the phosphorylation of consensus CDK phosphorylation sites as well as of nucleolin at a specific CDK1/cyclin B phosphorylation site, suggesting a direct action on the mitotic CDK1/cyclin B. When indirubin-3'-monoxime is added to HBL-100 cells synchronized in M phase by nocodazole, cells undergo an endoreplication leading to an 8n DNA content. As soon as indirubin-3'-monoxime is washed away, these polyploid cells become aneuploid and later die from necrosis. This mechanism of endoreplication followed by cell death may contribute to the anti-tumour properties of indirubins.

Antibiotics, Antineoplastic↗
PubMed2001

Inhibitor binding to active and inactive CDK2: the crystal structure of CDK2-cyclin A/indirubin-5-sulphonate.

BACKGROUND: Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2. RESULTS: Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten. CONCLUSIONS: The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.

CDC2-CDC28 Kinases↗
PubMed2001

[Analysis of symptoms associated with bovine herpesvirus 1 vaccination].

Between 1 May 1998 and 22 February 1999, it was compulsory for Dutch cattle farmers to take measures against bovine herpesvirus 1 (BHV1). Cattle on farms that were not certified as infectious bovine rhinotracheitis (IBR)-free had to be vaccinated twice a year. During the vaccination programme, both farmers and veterinarians reported side-effects of the vaccine. These reports were collected by the Stichting IBR/BVD Schade (SIS; Foundation for IBR/BVD Damage) in order to draw up a damage report. In 1999 in total 6977 cattle farmers lodged complaints which they considered to be related to the vaccination against BHV1. On these farms, 15,150 herd vaccinations had been performed, 10,269 of which were associated with one or more symptoms. During the compulsory vaccination period, 13% of the herd vaccinations led to symptoms and complaints. In March 1999, a number of vaccine batches were found to be contaminated with bovine virus diarrhoea (BVD) virus. For the purposes of this analysis, a 'known contaminated' herd vaccination was defined as one in which at least one 'known contaminated' batch or lot of vaccine was used. In total, 987 of 1007 herds vaccinated with 'known contaminated' vaccines developed one or more symptoms compatible with acute BVD. There were no commonly seen combinations of symptoms. For this reason, and because the start and end dates were not reported for 55% of the symptoms, it was not possible to detect a symptom pattern. Therefore there were no 'suspect' batches of vaccine which, although not contaminated with BVD virus, gave rise to symptoms. The number of BVD symptoms was determined for those herds with vaccination-related symptoms. There was no difference in the distribution frequency between batch numbers or between 'known contaminated' batches and 'non-suspect' batches. The farmers' definition of chronic wasting was used in this investigation, with the inevitable large differences in definition. The symptom chronic 'wasting' was reported for 3209 of the 10,269 herds with vaccination-related symptoms. On 161 farms (164 herd vaccinations) 'chronic wasting' accounted for more than 20% of the symptoms. As expected, other symptoms were reported in addition to wasting. The symptom 'chronic wasting' was reported more often on forms where a 'known contaminated' vaccine was used. Inactivated vaccine was used for 154 herd vaccinations. In 34 cases, one or more symptoms of acute BVD were reported. The frequency was the same as that for live vaccines. The frequency of reported symptoms tended to be lower with the inactivated vaccine. On the basis of the SIS data, no relationship was found between vaccine batch and reported symptoms. This may be because (i) the classification of a vaccine as 'known contaminated', 'non-suspect', and 'not known' may not have been in keeping with the real status of the vaccine, (ii) farmers may have reported symptoms selectively, and (iii) there is no relationship with vaccination against BHV1.

Animals↗
PubMed2001

Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.

Adenosine Triphosphate↗
PubMed2001

Coscinosulfate, a CDC25 phosphatase inhibitor from the sponge Coscinoderma mathewsi.

The dual specificity CDC25 phosphatases dephosphorylate two inhibitory phospho-amino acids of cyclin-dependent kinases, a major family of cell cycle regulators. CDC25 inhibitors constitute new anti-mitotic agents with potential anticancer activity. While screening through a collection of natural products derived from marine organisms for CDC25A inhibitors, we purified and identified coscinosulfate 1, a sesquiterpene sulfate from the New Caledonian sponge Coscinoderma matthewsi, along with 4. The purified compound 1 displayed significant inhibitory activity towards CDC25A (IC(50): 3 microM).

Animals↗
PubMed2001

Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum Biochemical properties and possible involvement in MAPK regulation.

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.

Amino Acid Sequence↗
PubMed2001

Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells.

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G(1)/G(0)phase of the cell cycle in the presence of 2 microM indirubin-3'-monoxime. At higher drug concentrations (> or = 5 microM) an increase of the cell population in the G(2)/M phase is additionally observed. Cells synchronized in G(2)/M phase by nocodazole remain arrested in the G(2)/M phase after release, in the presence of indirubin-3'-monoxime (> or =5 microM). After 24 h treatment with 10 microM indirubin-3'-monoxime a sub-G(2)peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy.

Antibiotics, Antineoplastic↗
PubMed2001

Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium.

BACKGROUND: The Toll protein in Drosophila regulates dorsal ventral patterning during embryogenesis, and participates in antibacterial and antifungal host defense. Mammalian homologues are termed Toll-like receptors and, to date, nine have been cloned (TLRI-9) in humans. They are characterized by extracellular leucine-rich repeats and a cytoplasmic domain similar to the interleukin 1 receptor. Both TLR2 and TLR4 recognize various bacterial cell wall components including lipopolysaccharide (LPS). This results in the activation of the NFkappaB pathway. Peripheral blood mononuclear cells (PBMCs) express both TLR2 and TLR4. The authors hypothesized that the expression of TLR 2 and TLR4 in human intestinal epithelial cells differs from PBMCs because of the abundance of LPS in the intestinal lumen. METHODS: Epithelial cells were isolated from Caco-2 cells, fetal gut explants, and small bowel resection specimens using Hanks/ethylenediamine tetraacetic acid solution. PBMCs were used as positive controls. Ribonucleic acid (RNA) was isolated using the TRIzol method. Standard reverse transcription-polymerase chain reaction examined TLR2 and TLR4 messenger RNA (mRNA) expression. NFkappaB expression was determined using a luciferase reporter assay. RESULTS: TLR2 mRNA was highly expressed in PBMCs and was present in all human intestinal epithelial cells. TLR4 mRNA was detected only in PBMCs. TLR4 is not present in epithelium from children with inflammatory bowel disease. In Caco-2 cells, significant NFkappaB activation in response to LPS occurred only in the presence of TLR4 introduced by complementary deoxyribonucleic acid transfection. CONCLUSION: Absence of TLR4 is associated with endotoxin hyporesponsiveness of intestinal epithelial cells. TLR4 is not directly involved in inflammation of the intestinal epithelium. Although TLR2 is normally present in the epithelial cell, it plays a limited role in inflammation. It may be activated during conditions in which bacterial cell wall concentrations within the intestine are pathologically high.

Adolescent↗
PubMed2001

In vitro activities of two antimitotic compounds, pancratistatin and 7-deoxynarciclasine, against Encephalitozoon intestinalis, a microsporidium causing infections in humans.

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC(50)s) of pancratistatin and 7-deoxynarciclasine for E. intestinalis were 0.18 microM and 0.2 microM, respectively, approximately eightfold lower than the IC(50)s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

Amaryllidaceae Alkaloids↗
PubMed2001

Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases (cdk's) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH(2), 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdk's. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G(1)/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdk's 9 and 7 was determined to be approximately 0.6 microM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdk's are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.

Cell Line↗
PubMed2001

[Prevention of chemotherapy-induced alopecia by cyclin-dependant kinase inhibitors].

Cyclin-dependent kinases (CDKs) are widely involved in regulating the cell division cycle. The discovery of numerous alterations of CDKs, cyclins, their regulators and their substrates in many human tumours has strongly stimulated the search for chemical inhibitors of CDKs. The potential use of these potent and selective kinase inhibitors in cancer therapy is presently under investigation. Recently a group from Glaxo Wellcome unexpectedly discovered a potential new application of these CDK inhibitors to prevent chemotherapy-induced alopecia. These cyto-protective properties might also be extended to other tissues damaged during chemotherapy.

Alopecia↗