Biomedical subjects

C Lukas

Publications and source records attributed to C Lukas.

11 recordsLinked to original sources

PubMed2006

EULAR recommendations for the management of early arthritis: report of a task force of the European Standing Committee for International Clinical Studies Including Therapeutics (ESCISIT).

OBJECTIVE: To formulate EULAR recommendations for the management of early arthritis. METHODS: In accordance with EULAR's "standardised operating procedures", the task force pursued an evidence based approach and an approach based on expert opinion. A steering group comprised of 14 rheumatologists representing 10 European countries. The group defined the focus of the process, the target population, and formulated an operational definition of "management". Each participant was invited to propose issues of interest regarding the management of early arthritis or early rheumatoid arthritis. Fifteen issues for further research were selected by use of a modified Delphi technique. A systematic literature search was carried out. Evidence was categorised according to usual guidelines. A set of draft recommendations was proposed on the basis of the research questions and the results of the literature search.. The strength of the recommendations was based on the category of evidence and expert opinion. RESULTS: 15 research questions, covering the entire spectrum of "management of early arthritis", were formulated for further research; and 284 studies were identified and evaluated. Twelve recommendations for the management of early arthritis were selected and presented with short sentences. The selected statements included recognition of arthritis, referral, diagnosis, prognosis, classification, and treatment of early arthritis (information, education, non-pharmacological interventions, pharmacological treatments, and monitoring of the disease process). On the basis of expert opinion, 11 items were identified as being important for future research. CONCLUSIONS: 12 key recommendations for the management of early arthritis or early rheumatoid arthritis were developed, based on evidence in the literature and expert consensus.

Antirheumatic Agents↗

PubMed2006

Repeat intrathecal triamcinolone acetonide application is beneficial in progressive MS patients.

Available immunomodulatory and conventional steroid treatment regimens provide a limited symptomatic benefit for patients with progressive multiple sclerosis (MS). We performed an open trial on the short-term efficacy of repeated intrathecal application of the sustained release steroid triamcinolone acetonide (TCA) in 27 progressive MS patients. Six TCA administrations, performed every third day, reduced the Expanded Disability Status Scale (EDSS) score [initial: 5.4+/-1.3, 3-7.5 (mean+/-SD, range); end: 4.9+/-1.1; 2.5-6.5; P<0.001] and significantly increased the walking distance and speed in particular after the fourth TCA injection. Concomitantly serially determined cerebrospinal fluid (CSF) markers of cell injury, neuron-specific enolase, total tau-protein, S-100, and beta-amyloid did not significantly change within the interval of TCA treatment. No serious side effects appeared. We conclude that repeat intrathecal injection of 40 mg TCA provides a substantial benefit in progressive MS patients with predominant spinal symptoms and does not alter CSF markers of neuronal cell injury.

Adult↗

PubMed2006

Oral fumaric acid esters for the treatment of active multiple sclerosis: an open-label, baseline-controlled pilot study.

An exploratory, prospective, open-label study of fumaric acid esters (FAE, Fumaderm(R)) was conducted in patients with relapsing-remitting multiple sclerosis (RRMS). The study consisted of the following four phases: 6-week baseline, 18-week treatment (target dose of 720 mg/day), 4-week washout, and a second 48-week treatment phase (target dose of 360 mg/day). Ten patients with an Expanded Disability Status Scale (EDSS) score of 2.0-6.0 and at least one gadolinium-enhancing (Gd+) lesion on T1-weighted magnetic resonance imaging (MRI) brain scans participated in the study. Safety was assessed by adverse events (AEs), blood chemistry/hematology, electrocardiogram, and urinalysis. The primary efficacy outcomes were number and volume of Gd+ lesions. Other clinical outcomes included EDSS score, ambulation index (AI), and nine-hole peg test (9-HPT). Effects of FAE on intracellular cytokine profiles, T-cell apoptosis, and soluble adhesion molecules were also assessed. Three patients withdrew during the first 3 weeks of the study because of side effects, non-compliance, and follow-up loss. The most common AEs were gastrointestinal symptoms and flushing; all AEs were reported as mild and reversible. FAE produced significant reductions from baseline in number (P < 0.05) and volume (P < 0.01) of Gd+ lesions after 18 weeks of treatment; this effect persisted during the second treatment phase at half the target dose after the 4-week washout period. EDSS scores, AI, and 9-HPT remained stable or slightly improved from baseline in all patients. Measures of T-cell function demonstrated alterations in cytokines and circulating tumor necrosis factor. The results of this exploratory study suggest that further studies of FAE in patients with MS are warranted.

Administration, Oral↗

PubMed2001

Functional impact of concomitant versus alternative defects in the Chk2-p53 tumour suppressor pathway.

Recent evidence identified a genetic and functional link between Chk2 kinase and p53 as a candidate genome integrity checkpoint and a tumour suppressor pathway. Here we report that in human cells, Chk2 and p53 form protein-protein complexes whose abundance increased upon DNA damage, and whose formation was abrogated through cancer associated mutations in the FHA domain of Chk2, or mutations in the tetramerization domain of p53. Whereas among Li-Fraumeni syndrome families mutations of Chk2 or p53 occur in a mutually exclusive manner, we document that the colon cancer cell line HCT-15 concomitantly lacks functions of both Chk2 and p53, the latter demonstrated by a non-invasive reporter assay monitoring p53-dependent transactivation in live cells. Despite the preserved ability of common cancer-derived mutant p53 proteins to bind and potentially 'titrate' activated Chk2, the integrity of the S phase checkpoint response to ionizing radiation remained largely intact and dependent on Chk2 in cells with wild-type, mutant, or no p53. These results provide new mechanistic insights into the Chk2-p53 interplay, suggest how mutations in Chk2 may abrogate its tumour suppressor function, and indicate that compared with individual defects in either Chk2 or p53, concomitant mutations in both of these cell cycle checkpoint regulators may provide some additional selective advantage to tumour cells.

Checkpoint Kinase 2↗

PubMed2001

DNA damage-activated kinase Chk2 is independent of proliferation or differentiation yet correlates with tissue biology.

The Chk2 kinase is a tumor suppressor and key transducer of DNA-damage checkpoints. We show that the human Chk2 protein is relatively stable, nuclear, and responding to gamma-radiation throughout the cell cycle. Contrary to the retinoblastoma protein-regulated, labile Chk1 kinase restricted to S-G(2) phases, Chk2 remains activatable even in quiescent and differentiating cells. In human tissues, Chk2 is homogeneously expressed in renewing cell populations such as epidermis or intestine, heterogeneous in conditionally renewing tissues, and absent or cytoplasmic in static tissues such as muscle or brain. These data highlight striking differences between Chk2 and Chk1 and show unexpected correlation of Chk2 expression with tissue biology.

Antibodies, Monoclonal↗

PubMed2001

A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression.

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

Amino Acid Sequence↗

PubMed2000

Rapid destruction of human Cdc25A in response to DNA damage.

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.

CDC2-CDC28 Kinases↗

PubMed2000

Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis.

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1999

Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex.

In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.

Anaphase↗

PubMed1999

p16INK4a, but not constitutively active pRb, can impose a sustained G1 arrest: molecular mechanisms and implications for oncogenesis.

p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.

Animals↗

PubMed1999

Immunohistochemical analysis of the D-type cyclin-dependent kinases Cdk4 and Cdk6, using a series of monoclonal antibodies.

Cellular signal transduction cascades triggered by mitogenic or antiproliferative cues eventually converge on a biochemical mechanism centered around the retinoblastoma tumor suppressor (pRb), the so-called RB pathway that governs G1-phase progression and guards the commitment to enter S phase. pRb, together with its immediate upstream regulators, the D-type cyclins, their partner cyclin-dependent kinases Cdk4 and Cdk6, and the Cdk inhibitors, form a functional unit that is involved in major decisions about cellular fate, and whose components, including the proto-oncogenic cyclin D-dependent kinases, are commonly deregulated in many types of cancer. We report here the production and characterization of a series of 12 monoclonal antibodies (MAbs) that specifically recognize either Cdk4 or Cdk6. These antibodies are proving to be invaluable molecular probes for defining abundance, subcellular localization, binding partners, and ultimately the function(s) of these cell cycle-regulatory kinases. Localization of the target epitopes was mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and two antibodies recognizing sequences adjacent to N-terminus of Cdk4 can discriminate between the wild-type protein and the oncogenic, melanoma-associated R24C mutant of this kinase. Individual antibodies of our panel recognize distinct pools of Cdk4/6, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, kinase assays, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Collectively, the antibodies described in this study provide the means for immunochemical analyses of the cyclin D-dependent kinases in human and animal cells, and represent useful molecular tools that should help better understand the biological roles of Cdk4 and Cdk6 in normal cell-cycle control, and their oncogenic activity in tumor cells.

Animals↗