Mitotic Spindle Checkpoint Research Articles

Histone H4 and the maintenance of genome integrity.

The normal progression of Saccharomyces cerevisiae through nuclear division requires the function of the amino-terminal domain of histone H4. Mutations that delete the domain, or alter 4 conserved lysine residues within the domain, cause a marked delay during the G2+M phases of the cell cycle. Site-directed mutagenesis of single and multiple lysine residues failed to map this phenotype to any particular site; the defect was only observed when all four lysines were mutated. Starting with a quadruple lysine-to-glutamine substitution allele, the insertion of a tripeptide containing a single extra lysine residue suppressed the G2+M cell cycle defect. Thus, the amino-terminal domain of histone H4 has novel genetic functions that depend on the presence of lysine per se, and not a specific primary peptide sequence. To determine the nature of this function, we examined H4 mutants that were also defective for G2/M checkpoint pathways. Disruption of the mitotic spindle checkpoint pathway had no effect on the phenotype of the histone amino-terminal domain mutant. However, disruption of RAD9, which is part of the pathway that monitors DNA integrity, caused precocious progression of the H4 mutant through nuclear division and increased cell death. These results indicate that the lysine-dependent function of histone H4 is required for the maintenance of genome integrity, and that DNA damage resulting from the loss of this function activates the RAD9-dependent G2/M checkpoint pathway.

Impact of oxaliplatin and a novel DACH-platinum complex in the gene expression of HCT116 colon cancer cells

Novel demethylcantharidin-platinum (DMC-Pt) complexes have been found to have superior in vitro anticancer activity against a number of human colon cancer cell lines when compared with oxaliplatin. One complex where the DMC-Pt moiety was integrated with trans-R,R-diamino-cyclohexane (DACH), exhibited the most pronounced cytotoxicity. To ascertain the mechanistic contribution of the DMC component, microarray analysis was conducted to compare the effect of the novel (R,R-DACH)-Pt-(DMC) complex and oxaliplatin, on the gene expression of human colorectal cancer (HCT116) cells. The Affymetrix HG-U133A oligonucleotide microarray was used, and the data allowed for the discrimination of genes that were specifically affected by the DMC ligand. One hundred and forty-one genes were found to be up-regulated. Of these, 48 can be classified according to different cellular responses including DNA repair, DNA synthesis, cell adhesion, cell cycle regulation, mitotic spindle checkpoint and apoptosis/antiapoptosis. The DMC ligand is likely to have caused damage to DNA bases and/or strands, and nucleotide mismatch, as highlighted by the recruitment of the repairing genes from the BER, HR and MMR. Antiapoptotic genes such as survivin, BRCA1 and ITGB3BP were up-regulated, and it is proposed that the inherent defense mechanism of the cell may have been triggered, creating potential resistance to apoptosis. This study is the first to demonstrate the impact of the DMC ligand on the gene expression profile of HCT116 colon cancer cells and further substantiates its inclusion in the design of novel platinum-based anticancer complexes.

The delta agent comes of age.

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent anti-mitotic components of natural origin but it is however intrinsically unstable. A novel series of CA-4 analogues incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesised with the objective to prevent cis-trans isomerisation and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogues demonstrated potent anti-tubulin, anti-proliferative and anti-mitotic effects in human leukemia cells. A lead β-lactam analogue, CA-476 displayed comparable antiproliferative activities with CA-4. CA-476 induced rapid apoptosis in HL-60 acute myeloid leukemia (AML) cells which was accompanied by depolymerisation of the microtubular network, PARP cleavage, caspase-3 activation and Bcl-2 cleavage. A prolonged G2M cell cycle arrest accompanied with a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the anti-apoptotic proteins Bcl-2 and Bcl-xL preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-476 induced significantly more apoptosis compared to imatinib mesylate in ex-vivo CML patient samples including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogues of CA-4 have been designed and synthesised which display significant clinical potential as anti-leukemic agents.