The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression

AbstractSiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.</jats:p

Aneuploidy in oocytes is prevented by sustained CDK1 activity through degron masking in cyclin B1

Successful mitosis requires that cyclin B1:CDK1 kinase activity remains high until chromosomes are correctly aligned on the mitotic spindle. It has therefore been unclear why, in mammalian oocyte meiosis, cyclin B1 destruction begins before chromosome alignment is complete. Here, we resolve this paradox and show that mouse oocytes exploit an imbalance in the ratio of cyclin B1 to CDK1 to control CDK1 activity; early cyclin B1 destruction reflects the loss of an excess of non-CDK1-bound cyclin B1 in late prometaphase, while CDK1-bound cyclin B1 is destroyed only during metaphase. The ordered destruction of the two forms of cyclin B1 is brought about by a previously unidentified motif that is accessible in free cyclin B1 but masked when cyclin B1 is in complex with CDK1. This protects the CDK1-bound fraction from destruction in prometaphase, ensuring a period of prolonged CDK1 activity sufficient to achieve optimal chromosome alignment and prevent aneuploidy

Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC 50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC 50 values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutic

Structure–activity relationship study of beta-carboline derivatives as haspin kinase inhibitors

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure–activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented

Structure–activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors

Haspin is a serine/threonine kinase required for completion of normal mitosis that is highly expressed during cell proliferation, including in a number of neoplasms. Consequently, it has emerged as a potential therapeutic target in oncology. A high throughput screen of approximately 140,000 compounds identified an acridine analog as a potent haspin kinase inhibitor. Profiling against a panel of 270 kinases revealed that the compound also exhibited potent inhibitory activity for DYRK2, another serine/threonine kinase. An optimization study of the acridine series revealed that the structure–activity relationship (SAR) of the acridine series for haspin and DYRK2 inhibition had many similarities. However, several structural differences were noted that allowed generation of a potent haspin kinase inhibitor (33, IC50 <60 nM) with 180-fold selectivity over DYRK2. In addition, a moderately potent DYRK2 inhibitor (41, IC50 <400 nM) with a 5.4-fold selectivity over haspin was also identified

Cadherin-11 Provides Specific Cellular Adhesion between Fibroblast-like Synoviocytes

Cadherins are integral membrane proteins expressed in tissue-restricted patterns that mediate homophilic intercellular adhesion. During development, they orchestrate tissue morphogenesis and, in the adult, they determine tissue integrity and architecture. The synovial lining is a condensation of fibroblast-like synoviocytes (FLS) and macrophages one to three cells thick. These cells are embedded within the extracellular matrix, but the structure is neither an epithelium nor an endothelium. Previously, the basis for organization of the synovium into a tissue was unknown. Here, we cloned cadherin-11 from human rheumatoid arthritis (RA)-derived FLS. We developed L cell transfectants expressing cadherin-11, cadherin-11 fusion proteins, and anti–cadherin-11 mAb. Cadherin-11 was found to be expressed mainly in the synovial lining by immunohistologic staining of human synovium. FLS adhered to cadherin-11–Fc, and transfection of cadherin-11 conferred the formation of tissue-like sheets and lining-like structures upon fibroblasts in vitro. These findings support a key role for cadherin-11 in the specific adhesion of FLS and in synovial tissue organization and behavior in health and RA

Protein engineering of human properdin

Properdin is a serum glycoprotein that upregulates the alternative pathway of complement by stabilizing the C3bBb complex. It also binds sulphated glycoconjugates, such as sulphatide, in vitro. Properdin is composed of cyclic dimers, trimers and tetramers of a 53 kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately sixty amino acids. The sixth TSR of properdin contains an insertion of approximately 30 amino acids which corresponds to the position of an intron in the human properdin gene. In order to identify the regions of properdin important for function, human properdin, and mutant forms each lacking a single TSR, were expressed in Chinese Hamster Ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of the normal and altered forms of properdin were investigated. Wild type recombinant properdin is similar to properdin purified from plasma in size, immunoreactivity, N-terminal sequence, possession of N-linked sugar, oligomerization (as determined by electron microscopy and gel exclusion chromatography), and functional activity in an alternative pathway haemolytic assay, and in C3b and sulphatide binding assays. Properdin "nicked" in TSR5 is unable to bind C3b, while retaining its overall structure and its ability to bind sulphatide. The removal of TSRS prevents C3b and sulphatide binding. Properdin lacking TSR4 is unable to stabilize the C3bBb complex, but is able to bind C3b and sulphatide, and shows the presence of monomers and dimers in the electron microscope. Properdin without TSR3 is able to stabilize the C3bBb complex, to bind CSb and sulphatide, and forms dimers, trimers and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3bBb complex. Monoclonal antibodies which bind to the N-terminal region, TSR1, or TSR2 are able to inhibit properdin binding to CSb. A monoclonal antibody which binds TSR4 is able to inhibit properdin binding to sulphatide, but not to CSb. The results confirm that TSRs are folded as independent units. The N-terminal end and TSR5 of properdin are implicated in CSb binding. The vertices of properdin oligomers may be important for interaction with CSb. TSR4 may also be involved in stabilization of the C3bBb complex. The sulphatide binding site is distinct from the CSb binding site, but TSR5, which contains many basic residues, may be important for both activities.</p