Abstract
Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.
Highlights
In normal cell division, centrosomes undergo one round of duplication in a manner analogous to the replication of chromosomal DNA during S phase (4)
The first 18 residues of the ECRG2 protein are a signal peptide to mediate targeting to the endoplasmic reticulum for secretion, pulse-chase experiments showed that ECRG2 was transferred to both intracellular and extracellular cells
ECRG2 accumulated around kinetochores
Summary
Generation of ECRG2 Antibody—We generated anti-ECRG2 monoclonal antibodies by using the C-terminal of ECRG2 peptide (KSNGRVQFLHDGSC) as immunogen (28). 217–258 encoding portions of human ECRG2 were amplified using Pfu polymerase (Stratagene), cloned into pGEX-4T-3 (GE Healthcare) as BamHI/NotI restriction fragments. After each chase period the supernatants were harvested and the cells were lysed Both the supernatants and cell lysates were incubated with antibodies against ECRG2 or normal IgG (control) for 2 h at 4 °C, followed by incubation with protein A-agarose beads for an additional 2 h at 4 °C. The beads were pelleted, washed extensively with immunoprecipitation buffer, boiled in SDS sample buffer, fractionated by SDS-PAGE, and analyzed by anti-ECRG2 or p53 antibody. For isolation of CDC2 or CDK2 complexes, immunoprecipitation assay were performed by incubating 500 g of total proteins from cell lysates described above or ECRG23ЈUTR-KD-Tet-on cell lysates with rabbit polyclonal anti-CDC2 or anti-CDK2 antibodies (Santa Cruz) for 2 h at 4 °C, respectively. The eluted proteins were analyzed by Western blot for the presence of conjugated p53 by DO-1 antibody
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