These results indicate that the reduction of CENP-A drives normal human diploid fibroblasts into a senescent state in a p53-dependent manner

These results indicate that the reduction of CENP-A drives normal human diploid fibroblasts into a senescent state in a p53-dependent manner. damage, oxidative stress, telomere shortening, and oncogene activation (7, 14, 15, 25, 55). While senescent cells Roburic acid maintain metabolic activity, cell cycle progression is Roburic acid permanently inhibited. The molecular basis of senescence has been studied intensively using normal diploid fibroblasts, melanocytes, and epithelial cells. In these studies, two tumor suppressor molecules, p53 and retinoblastoma protein (Rb), have been shown to play crucial roles in cell cycle arrest in senescent cells. In these cells, p53 effectively blocks cell cycle progression by upregulating its transcriptional target, p21CIP1. Rb is activated by p21CIP1 and p16INK4a, both of which are highly expressed in senescent cells (1, 7, 24). Activated Rb binds to E2F transcription factors to repress the expression of E2F target genes that promote cell proliferation (34). In contrast, p53 and p16INK4a-Rb pathways are often mutated in tumors (26, 53), and such tumor cells keep growing indefinitely without ever entering a senescent state. Senescence is therefore presumed to be a self-defense mechanism that prevents the uncontrolled proliferation of tumorigenic cells. Although it remains to be established how senescence, including the activation of the tumor suppressors, is initiated, certain defects in chromosome integrity, such as telomere shortening, can trigger it (7, 15). It was recently reported that BubR1-insufficient and Bub3/Rae1-haploinsufficient mice display an array of early aging-associated phenotypes (3-5) and Bub1 suppression in human fibroblasts activates a p53-dependent premature senescence response (22). Bub1, BubR1, and Bub3 are key players in the spindle assembly checkpoint (SAC) that blocks mitotic progression into anaphase in response to abnormalities in kinetochore-spindle interaction and/or kinetochore structure. These observations suggest that, like telomeres, kinetochores may also play Roburic acid a crucial role in regulating commitment to the senescent state. Kinetochores are multiprotein complexes formed on a specialized region of each chromosome, designated the centromere. Kinetochore function is essential for the faithful segregation of chromosomes during mitosis and meiosis (13, 40). The centromere is composed of two domains, core centromeric chromatin and pericentric heterochromatin region. Numerous kinetochore-associated proteins have been identified to date, including centromere proteins (CENPs), Mis12, and SAC proteins (20, Roburic acid 23, 29, 40, 45, 47). CENP-A is an evolutionarily conserved centromere-specific histone H3 variant (8, 11, 18, 38, 49, 57, 59). As such, CENP-A represents an excellent candidate for an epigenetic marker of functional centromeres that could be monitored by senescence promoting networks. Studies of a variety of organisms have indicated that CENP-A plays a crucial role in organizing kinetochore chromatin for precise chromosome segregation; however, the impact of CENP-A loss upon proliferation varies widely in the context of species, cell types, and methods used to delete or deplete CENP-A (8, 23, 27, 51). CENP-B is another conserved centromere protein. CENP-B binds to a specific centromeric DNA sequence, the 17-bp CENP-B box in type I -satellite repeats in human cells (19, 36). CENP-B is also important for proper organization of kinetochore chromatin. Although CENP-B is not essential for viability in higher eukaryotes (28, 30, 50), it is essential for heterochromatin formation of pericentromeres (41, 42, 48). Despite the extensive studies of centromere-associated proteins, it remains unclear whether these proteins are involved in the control of cell proliferation; previous studies focused on Rabbit Polyclonal to LAMA5 the roles of centromere proteins in chromosome segregation and were mainly conducted in immortalized cell lines, such as Roburic acid HeLa. In HeLa cells, p53 and Rb are known to be inactivated due to the integration of the human papillomavirus that leads to their immortalization. Although it is essential to use primary human cells to uncover the regulatory roles of centromere proteins in cell proliferation, such studies have not been done. In our exploration of senescence-associated alterations in nuclear structure using primary human cells, we found that CENP-A levels were markedly reduced in the senescent cells. Furthermore, we showed that short-hairpin RNA (shRNA)-mediated depletion of CENP-A induces senescence in primary human fibroblast TIG3 cells but not HeLa cells. Inactivation of p53 in TIG3 cells depleted of CENP-A restores the proliferation, leading to an increase in the number of cells exhibiting aberrant chromosome behavior. These results indicate that the reduction of CENP-A drives normal human diploid fibroblasts into a senescent state in a p53-dependent manner. The senescence that arises from CENP-A depletion may be a self-defense mechanism to suppress.