Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

CRMP4 CpG Hypermethylation Predicts Upgrading to Gleason Score ≥ 8 in Prostate Cancer.

Background: This study determined the predictive value of CRMP4 promoter methylation in prostate tissues collected by core needle biopsies for a postoperative upgrade of Gleason Score (GS) to ≥8 in patients with low-risk PCa. Method: A retrospective analysis of the clinical data was conducted from 631 patients diagnosed with low-risk PCa by core needle biopsy at multiple centers and then underwent Radical Prostatectomy (RP) from 2014-2019. Specimens were collected by core needle biopsy to detect CRMP4 promoter methylation. The pathologic factors correlated with the postoperative GS upgrade to ≥8 were analyzed by logistic regression. The cut-off value for CRMP4 promoter methylation in the prostate tissues collected by core needle biopsy was estimated from the ROC curve in patients with a postoperative GS upgrade to ≥8. Result: Multivariate logistic regression showed that prostate volume, number of positive cores, and CRMP4 promoter methylation were predictive factors for a GS upgrade to ≥8 (OR: 0.94, 95% CI: 0.91-0.98, P=0.003; OR: 3.16, 95% CI: 1.81-5.53, P<0.001; and OR: 1.43, 95% CI: 1.32-1.55, P<0.001, respectively). The positive predictive rate was 85.2%, the negative predictive rate was 99.3%, and the overall predictive rate was 97.9%. When the CRMP4 promoter methylation rate was >18.00%, the low-risk PCa patients were more likely to escalate to high-risk patients. The predictive sensitivity and specificity were 86.9% and 98.8%, respectively. The area under the ROC curve (AUC) was 0.929 (95% CI: 0.883-0.976; P<0.001). The biochemical recurrence (BCR)-free survival, progression-free survival (PFS), and cancer-specific survival (CSS) were worse in patients with CRMP4 methylation >18.0% and postoperative GS upgrade to ≥8 than in patients without an upgrade (P ≤ 0.002). Conclusion: A CRMP4 promoter methylation rate >18.00% in prostate cancer tissues indicated that patients were more likely to escalate from low-to-high risk after undergoing an RP. We recommend determining CRMP4 promoter methylation before RP for low-risk PCa patients.

Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice.

1. 2-p-(2-carboxyethylphenethylamino-5'-ethylcarboxamidoadenosine) (CGS 21680) is considered the reference compound to study adenosine A(2A) receptors. However, CGS 21680 binding in the cerebral cortex, where adenosine A(1) receptors are predominant, displays a mixed A(2A)/A(1) receptor pharmacology. We now use adenosine A(1) and A(2A) receptor knockout mice to investigate the characteristics of cortical [(3)H]CGS 21680 binding. 2. [(3)H]CGS 21680 binding to the cerebral cortex was strongly reduced in adenosine A(1) receptor knockout mice, but only slightly reduced in A(2A) receptor knockout mice compared with the corresponding wild-type littermates. 3. Another selective A(2A) receptor ligand, [(3)H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), displayed a saturable binding to mouse cortical membranes, albeit with a binding density 20 times lower than that of striatal membranes, and this [(3)H]SCH58261 binding was abolished in both striatal and cortical membranes of A(2A) receptor knockout mice and unchanged in A(1) receptor knockout mice. 4. The presence of A(2A) receptors in cortical neurons was further confirmed by Western blot in mouse cortical nerve terminal membranes. 5. It is concluded that, although A(2A) receptors are present in the cerebral cortex, the purportedly selective A(2A) receptor agonist [(3)H]CGS 21680 binds in the cerebral cortex to an entity that requires the presence of adenosine A(1) receptors. Thus, CGS 21680 should be used with care in all preparations where adenosine A(1) receptors out-number A(2A) receptors.