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Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.

ABSTRACT: Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G(2) checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G(2) checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks.

SUBMITTER: Zhao H

PROVIDER: S-EPMC137498 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.

Zhao Hui H Watkins Janis L JL Piwnica-Worms Helen H

Proceedings of the National Academy of Sciences of the United States of America 20021024 23

Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G(2) checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A ...[more]

PMID: 12399544

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Project description:BackgroundMajor genomic surveillance mechanisms regulated in response to DNA damage exist at the G1/S and G2/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.ResultsUsing an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.ConclusionOverall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.

Project description:Long non?coding RNA (lncRNA) LINC00473 plays a carcinogenic role in a variety of different tumor types. Nevertheless, the mechanisms through which LINC00473 regulates the radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells remains elusive. In the present study, reverse transcription?quantitative PCR was used to quantify the expression of LINC00473, microRNA (miRNA/miR)?497?5p and cell division cycle 25A (CDC25A) in ESCC tissues. The association between LINC00473 expression and the clinicopathological characteristics of patients with ESCC was also assessed. Furthermore, Cell Counting kit?8 and colony formation assays were carried out to monitor the proliferation of ESCC cells exposed to X?ray radiation. A dual?luciferase reporter assay was also conducted to analyze the interaction between LINC00473 and miR?497?5p, as well as the interaction between CDC25A and miR?497?5p. The findings of the present study demonstrated that in ESCC tissues and cells, the expression levels of LINC00473 and CDC25A were significantly upregulated, while the expression of miR?497?5p was downregulated. The high expression level of LINC00473 was associated with a higher T stage, lymph node metastasis stage and a lower tumor differentiation grade in patients with ESCC. Following irradiation, transfection with miR?497?5p mimics reduced the promoting effect of LINC00473 overexpression on ESCC cell proliferation, and partially impeded the resistance of ESCC cells to X?ray radiation induced by LINC00473 overexpression. Moreover, transfection with miR?497?5p inhibitors partially alleviated the inhibitory effects of LINC00473 knockdown on cellular proliferation, and partly reversed the sensitivity of cells to X?ray irradiation induced by LINC00473 knockdown. Furthermore, it was confirmed that miR?497?5p was able to bind LINC00473 and the 3'?untranslated region of CDC25A. On the whole, the findings of the present study demonstrate that LINC00473 reduces the radiosensitivity of ESCC cells by modulating the miR?497?5p/CDC25A axis.

Dataset Information

Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.

Publications

Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints.

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