Abstract
Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.
Highlights
Cells are continuously faced with exogenous and endogenous stress that can induce DNA damage, potentially leading to genomic instability and cell death [1]
Chemicals and antibodies Cisplatin was purchased from Sigma; protein associated splicing factor (PSF) and p54nrb antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal anti-b-actin antibody and the Annexin V-fluoresce isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit were obtained from Multisciences Biotechnology (Hangzhou, China). cH2AX, Rad51 and 53BP1 antibodies were purchased from Millipore (Billerica, MA); Caspase-3 and PARP antibodies were supplied by Bioworld Technology
To further validate this observation, Human cervical carcinoma (HeLa) cells were treated with different doses of cisplatin for 12 h, and the expression of Paraspeckle protein 1 (PSPC1) was examined by Western blot
Summary
Cells are continuously faced with exogenous and endogenous stress that can induce DNA damage, potentially leading to genomic instability and cell death [1]. DNA damage is primarily detected by the MRE11–RAD50–NBS1 (MRN) complex, which is followed by the activation of the phosphatidylinositol 3-kinase-like protein kinase (PIKKs) family members: ataxia telangiectasia mutated protein (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and DNA dependent protein kinase (DNA-PK) [2,3,4] These kinases phosphorylate and activate a variety of substrates to execute various cellular functions such as DNA repair, cell cycle arrest and cell death. Phosphorylation of H2AX is required to recruit a number of DDR proteins including repair factors and chromatin remodeling complexes [7,8,9] For this reason, cH2AX foci formation has been recognized as an effective indicator of DNA damage, even when only a few DNA double-strand breaks (DSBs) are elicited [10,11,12]. Defective DNA damage repair is associated with various developmental, immunological, and neurological disorders, and is a major driver in cancer [20]
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