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PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide.

ABSTRACT: A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies.

SUBMITTER: Hoang-Minh LB

PROVIDER: S-EPMC5035360 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide.

Hoang-Minh Lan B LB Deleyrolle Loic P LP Nakamura Nariaki S NS Parker Alexander K AK Martuscello Regina T RT Reynolds Brent A BA Sarkisian Matthew R MR

Translational oncology 20160920 5

A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agent ...[more]

PMID: 27661404

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Project description:Temozolomide (TMZ) is an alkylating agent used in the treatment of high-grade malignant glioma, notably glioblastoma multiforme, the most aggressive form of brain cancer. The drug induces a dozen DNA methylation adducts, including O⁶-methylguanine (O⁶MeG), which is the most toxic primary DNA lesion as it causes the formation of DNA double-strand breaks (DSBs) that trigger apoptosis. In p53 wild-type cells, TMZ activates p-p53ser15 and p-p53ser46, which have opposing dual functions regulating survival and death, respectively. Since the use of TMZ in a therapeutic setting is limited because of its side effects, the question arises as to the existence of threshold doses that activate the death pathway and start apoptosis. To determine whether there is a threshold for the TMZ-induced DNA damage response and exploring the factors regulating the switch between p53 dependent survival and death, the glioblastoma lines LN-229 (deficient in MGMT) and LN-229MGMT (stably transfected with MGMT) were exposed to different doses of TMZ. p53 protein expression and phosphorylation levels of p-p53ser15 and p-p53ser46 were determined by Western blotting. Also, apoptosis, senescence and autophagy levels were checked after different doses of TMZ. The results show that pro-survival p-p53ser15 and pro-death p-p53ser46 were induced by O⁶MeG in a specific dose- and time-dependent manner. p-p53ser15 was an early response while p-p53ser46 was activated at later times following treatment. Unexpectedly, the dose-response curves for total p53, p-p53ser15 and p-p53ser46 were linear, without an obvious threshold. O⁶MeG induces apoptosis late after treatment as a linear function of TMZ dose. This was observed for both p53 proficient LN-229 and p53 lacking LN-308 cells. A linear dose-response after TMZ was also observed for senescence and autophagy as well as γH2AX, an indicator of DSBs that are considered to be the downstream trigger of apoptosis, senescence and autophagy. LN-229MGMT cells were highly resistant to all measured endpoints because of repair of the critical primary lesion. Although LN-308 were less responsive than LN-229 to TMZ, they displayed the same TMZ-induced DSB level. The observed linear dose-responses are not compatible with the view that low DNA damage level evokes survival while high damage level activates death functions. The data bear important therapeutic implications as they indicate that even low doses of TMZ may elicit a cytotoxic response. However, since O⁶MeG triggers apoptosis, senescence and autophagy in the same dose range, it is likely that the accumulation of senescent cells in the population counteracts the killing effect of the anticancer drug.

Dataset Information

PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide.

Publications

PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide.

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