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MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR-301b-3p functions as a driver in various types of human cancer. However, the expression pattern of miR-301b-3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR-301b-3p expression was significantly up-regulated in HCC tissues compared to adjacent non-tumour tissues. Clinical association analysis revealed that the high level of miR-301b-3p closely correlated with large tumour size and advanced tumour-node-metastasis stages. Importantly, the high miR-301b-3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR-301b-3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR-301b-3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR-301b-3p directly bond to 3'UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down-regulated and inversely correlated with miR-301b-3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR-301b-3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR-301b-3p is highly expressed in HCC. miR-301b-3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Checkpoint kinase 1 (CHK1) is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumor cells have developed a strong dependence on CHK1 for survival, and hence, this kinase has developed into a promising drug target. Chk1 deficiency in mice results in blastocyst death due to G2/M checkpoint failure showing that it is an essential gene and may be difficult to target therapeutically. Here, we show that chemical inhibition of CHK1 kills murine and human hematopoietic stem and progenitor cells (HSPCs) by the induction of BCL2-regulated apoptosis. Cell death in HSPCs is independent of p53 but requires the BH3-only proteins BIM, PUMA, and NOXA. Moreover, Chk1 is essential for definitive hematopoiesis in the embryo. Noteworthy, cell death inhibition in HSPCs cannot restore blood cell formation as HSPCs lacking CHK1 accumulate DNA damage and stop dividing. Moreover, conditional deletion of Chk1 in hematopoietic cells of adult mice selects for blood cells retaining CHK1, suggesting an essential role in maintaining functional hematopoiesis. Our findings establish a previously unrecognized role for CHK1 in establishing and maintaining hematopoiesis., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

In this study, we explored the therapeutic potential of microRNA (miR) analogs against non-small-cell lung cancer (NSCLC) using lentiviral delivery of short hairpin RNA (shRNA). By using A549 as a model cell line, we used analogs and mimics of miR-4319/miR-125-5p to target the tumorigenic RAF1 gene. Lentiviral vectors carrying shRNA of a highly efficient miRNA analog of miR-4319/miR-125-5p, Analog2, were constructed to infect A549 cells. Our results showed that, compared with the noncancerous bronchial epithelial cell line 16HBE, lentivirus delivering Analog2 shRNA induced significant G2/M arrest and subsequent apoptosis in A549 cells, but not in 16HBE cells. Western blot analysis revealed that key factors regulating cell cycle were downregulated following RAF1 inhibition. In vivo xenograft experiments showed that lentivirus carrying Analog2 shRNA markedly decreased tumor size. Therefore, lentiviral delivery of Analog2 shRNA is a valid RNA interference-based treatment against NSCLC with high potency and specificity., (© 2019 Wiley Periodicals, Inc.)

Diallyl disulfide (DADs), a natural organic compound, is extracted from garlic and scallion and has anti-tumor effects against various tumors. This study investigated the anti-tumor activity of DADs in human osteosarcoma cells and the mechanisms. MG-63 cells were exposed to DADs (0, 20, 40, 60, 80, and 100 μM) for different lengths of time (24, 48, and 72 h). The CCK8 assay results showed that DADs inhibited osteosarcoma cell viability in a dose-and time-dependent manner. FITC-Annexin V/propidium iodide staining and flow cytometry demonstrated that the apoptotic ratio increased and the cell cycle was arrested at the G 2 /M phase as the DADs concentration was increased. A Western blot analysis was employed to detect the levels of caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and p62 as well as suppression of the mTOR pathway. High expression of LC3-II protein revealed that DADs induced formation of autophagosome. Furthermore, DADs-induced apoptosis was weakened after adding 3-methyladenine, demonstrating that the DADs treatment resulted in autophagy-mediated death of MG-63 cells. In addition, DADs depressed p-mTOR kinase activity, and the inhibited PI3K/Akt/mTOR pathway increased DADs-induced apoptosis and autophagy. In conclusion, our results reveal that DADs induced G 2 /M arrest, apoptosis, and autophagic death of human osteosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

Malignant melanoma is the most lethal type of skin cancer. Previous studies have shown that ailanthone has potent antitumor activity in a variety of cell lines. However, the anti-tumor effect of ailanthone on malignant melanoma remains unclear. To investigate the anti-tumor mechanisms of ailanthone in human melanoma B16 and mouse melanoma A375 cells, the cell counting kit-8 assay, colony formation assay, DNA content analysis, Hoechst 33258, and Annexin V-FITC/PI staining were used to assess cell proliferation, cell cycle distribution, and cell apoptosis, respectively. Western blotting was performed to evaluate the expression of cell cycle- and apoptosis-related proteins and regulatory molecules. The results showed that ailanthone significantly inhibited melanoma B16 and A375 cell proliferation as well as remarkably induced cell cycle arrest at the G0-G1 phase in B16 cells and the G2-M phase in A375 cells in a dose-dependent manner. Further investigation revealed that ailanthone promoted the expression of p21 and suppressed the expression of cyclin E in B16 cells or cyclin B in A375 cells through the PI3K-Akt signaling pathway. In addition, ailanthone induced B16 and A375 cell apoptosis via a caspase-dependent mechanism. Further studies showed that ailanthone remarkably downregulated Bcl-2 and upregulated Apaf-1 and Bax, and subsequently increased mitochondrial membrane permeabilization and released cytochrome c from the mitochondria in B16 cells and A375 cells. Taken together, ailanthone induces cell cycle arrest via the PI3K-Akt signaling pathway as well as cell apoptosis via the mitochondria-mediated apoptotic signaling pathway. Ailanthone may be potentially utilized as an anti-tumor agent in the management of malignant melanoma.

Long-term sequelae of acute kidney injury (AKI) are associated with incomplete recovery of renal function and the development of chronic kidney disease (CKD), which can be mediated by aberrant innate immune activation, mitochondrial pathology, and accumulation of senescent tubular epithelial cells (TECs). Herein, we show that the innate immune receptor Triggering receptor expressed on myeloid cells-1 (TREM-1) links mitochondrial metabolism to tubular epithelial senescence. TREM-1 is expressed by inflammatory and epithelial cells, both players in renal repair after ischemia/reperfusion (IR)-induced AKI. Hence, we subjected WT and TREM1/3 KO mice to different models of renal IR. TREM1/3 KO mice displayed no major differences during the acute phase of injury, but increased mortality was observed in the recovery phase. This detrimental effect was associated with maladaptive repair, characterized by persistent tubular damage, inflammation, fibrosis, and TEC senescence. In vitro , we observed an altered mitochondrial homeostasis and cellular metabolism in TREM1/3 KO primary TECs. This was associated with G2/M arrest and increased ROS accumulation. Further exposure of cells to ROS-generating triggers drove the cells into a stress-induced senescent state, resulting in decreased wound healing capacity. Treatment with a mitochondria anti-oxidant partly prevented the senescent phenotype, suggesting a role for mitochondria herein. In summary, we have unraveled a novel (metabolic) mechanism by which TREM1/3 deficiency drives senescence in TECs. This involves redox imbalance, mitochondrial dysfunction and a decline in cellular metabolic activities. These finding suggest a novel role for TREM-1 in maintaining tubular homeostasis through regulation of mitochondrial metabolic flexibility.

Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, we show that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death. We found that ERK is also activated in normal tissues by an exogenous source of damage and that this activation is necessary to dampen the pro-apoptotic role of Dp53. We exploit the pro-survival role of ERK activation upon endogenous and exogenous sources of DNA damage to present evidence that its genetic or chemical inhibition can be used as a therapeutic opportunity to selectively eliminate RAS-malignant tissues., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Resveratrol (RSV) has broad prospective applications as a radiation protection drug, but its mechanism of action is not yet clear. Here, we found that 5 μM RSV can effectively reduce the cell death caused by irradiation. Irradiation leads to G 2 /M phase arrest in the cell cycle, whereas RSV treatment increases S-phase cell cycle arrest, which is associated with sirtuin 1 (SIRT1) regulation. Meanwhile, RSV promotes DNA damage repair, mainly by accelerating the efficiency of homologous recombination repair. Under oxidative stress, tyrosyl-tRNA synthetase (TyrRS) is transported to the nucleus to protect against DNA damage. RSV can promote TyrRS acetylation, thus promoting TyrRS to enter the nucleus, where it regulates the relevant signaling proteins and reduces apoptosis and DNA damage. SIRT1 is a deacetylase, and SIRT1 knockdown or inhibition can increase TyrRS acetylation levels, further reducing radiation-induced apoptosis after RSV treatment. Our study revealed a new radiation protection mechanism for RSV, in which the acetylation of TyrRS and its translocation into the nucleus is promoted, and this mechanism may also represent a novel protective target against irradiation.-Gao, P., Li, N., Ji, K., Wang, Y., Xu, C., Liu, Y., Wang, Q., Wang, J., He, N., Sun, Z., Du, L., Liu, Q. Resveratrol targets TyrRS acetylation to protect against radiation-induced damage.

Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents re-replication in vertebrates and invertebrates. Previously, we identified two Geminin genes, BmGeminin1 and BmGeminn2 , in the silkworm Bombyx mori , and we found that RNA interference of BmGeminin1 led to re-replication. However, the function of BmGeminin2 remains poorly understood. In this study, we found that knockdown of BmGeminin2 can improve cell proliferation, and upregulated G2/M-associated gene- cyclinB / CDK1 expression. Then, we performed yeast two-hybrid screening to identify interacting proteins. Our results yielded 23 interacting proteins, which are involved in DNA replication, chromosome stabilization, embryonic development, energy, defense, protein processing, or structural protein. Here, we focused on BmRRS1, a chromosome congression-related protein that is closely related to cell cycle G2/M progression. The interaction between BmGeminin2 and BmRRS1 was confirmed by immunofluorescence and immunoprecipitation. Analysis of its expression profile showed that BmRRS1 was related to BmGeminin2 . In addition, BmGeminin2 overexpression downregulated the BmRRS1 transcript. Knockdown of BmGeminin2 led to upregulation of the BmRRS1 transcript. Furthermore, overexpression of BmRRS1 can upregulate G2/M-associated gene- cyclinB / CDK1 expression, and improved cell proliferation, consistent with the effects of BmGeminin2 knockout. In addition, BmRRS1 RNA interference can eliminate the impact of BmGem2 knockout on cell proliferation, the ratio of cell cycle stage and the expression of cyclinB / CDK1 . These data suggested that the cell proliferation advantage of BmGeminin2 knockout was closely related to BmRRS1 . Our findings provide insight into the functions of Geminin and the mechanisms underlying the regulation of the cell cycle in the silkworm.

Diacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, but its biological function on acute myeloid leukemia (AML) has not been explored. The aim of this study was to analyze apoptosis induced by knockdown of DGKZ and its mechanism in human acute myeloid leukemia HL-60 cells. qRT-PCR was carried out to detect the expression of DGKZ in HL-60, THP-1, Jurkat, K562, and CD34 cell lines. Additionally the expression of DGKZ in AML cells obtained from patients were detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of HL-60 cells DGKZ knocked down. Apoptosis and cell cycle phase of HL-60 cells after DGKZ knockdown were evaluated by flow cytometry. Western blot analysis was performed to investigate expressions of the proteins related to apoptosis and cell cycle. Results showed that expression of DGKZ was significantly higher in HL-60 and AML cells obtained from patients than those of Jurkat, THP-1, K562 and human CD34 cell. Compared with the shCtrl group, DGKZ was markedly knocked down in HL-60 cells transfected with lentivirus encoding shRNA. DGKZ knockdown significantly inhibited the proliferation and induced cycle arrest at the G2/M phase in HL-60 cells. The expressions of MAPK, caspase-3, caspase-8, cytochrome C markedly increased and p-MAPK and survivin decreased in HL-60 cells after DGKZ knockdown. The results suggest that knockdown of DGKZ can induce apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells through the MAPK/survivin/caspase pathway.

Staphylococcal nuclease and tudor domain containing 1 (SND1) has multiple functions in a variety of cellular processes. Here, we assessed the effects of SND1 in cellular DNA damage after ionizing radiation (IR). Knocking down SND1 in the mouse-derived photoreceptor 661 W cell line markedly inhibited cell proliferation and increased apoptosis after IR treatment. After DNA damage, SND1 induced Ataxia telangiectasia mutated kinase (ATM) signaling to launch DNA repair. Defects of SND1 were associated with missing response to DNA damage signal to cell cycle checkpoints or DNA repair. The current findings reveal SND1 as a new regulatory factor in DNA damage response., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Using data generated with cells exposed to ionizing-radiation (IR) in G 2 -phase of the cell cycle, we describe dose-dependent interactions between ATM, ATR and DNA-PKcs revealing unknown mechanistic underpinnings for two key facets of the DNA damage response: DSB end-resection and G 2 -checkpoint activation. At low IR-doses that induce low DSB-numbers in the genome, ATM and ATR regulate epistatically the G 2 -checkpoint, with ATR at the output-node, interfacing with the cell-cycle predominantly through Chk1. Strikingly, at low IR-doses, ATM and ATR epistatically regulate also resection, and inhibition of either activity fully suppresses resection. At high IR-doses that induce high DSB-numbers in the genome, the tight ATM/ATR coupling relaxes and independent outputs to G 2 -checkpoint and resection occur. Consequently, both kinases must be inhibited to fully suppress checkpoint activation and resection. DNA-PKcs integrates to the ATM/ATR module by regulating resection at all IR-doses, with defects in DNA-PKcs causing hyper-resection and G 2 -checkpoint hyper-activation. Notably, hyper-resection is absent from other c-NHEJ mutants. Thus, DNA-PKcs specifically regulates resection and adjusts the activation of the ATM/ATR module. We propose that selected DSBs are shepherd by DNA-PKcs from c-NHEJ to resection-dependent pathways for processing under the regulatory supervision of the ATM/ATR module.

Lysine-specific demethylase 1 (LSD1) is a histone modifier that is highly overexpressed in lung adenocarcinoma, which results in aggressive tumor biology. Tumor cell proliferation and migration analysis after LSD1 inhibition in the lung adenocarcinoma cell line PC9, using the LSD1 inhibitor HCI-2509 and siRNA, demonstrated that LSD1 activity was essential for proliferation and migration capacities of tumor cells. Moreover, reduced proliferation rates after LSD1 inhibition were shown to be associated with a cell-cycle arrest of the tumor cells in the G 2 -M-phase. Expression profiling followed by functional classification and pathway analysis indicated prominent repression of the polo-like kinase 1 (PLK1) pathway upon LSD1 inhibition. In contrast, transient overexpression of exogenous PLK1 plasmid rescued the LSD1 inhibition-mediated downregulation of PLK1 pathway genes. Mechanistically, LSD1 directly regulates expression of PLK1 by binding to its promoter region that subsequently affects expression of its downstream target genes. Notably, using lung adenocarcinoma TCGA datasets a significant correlation between LSD1 and PLK1 along with its downstream targets was observed. Furthermore, the LSD1/PLK1 linkage was confirmed by IHC analysis in a clinical lung adenocarcinoma cohort ( n = 43). Conclusively, this is the first study showing a direct transcriptional link between LSD1 and PLK1. IMPLICATIONS: These findings point to a role of LSD1 in regulating PLK1 and thus efficient G 2 -M-transition-mediating proliferation of tumor cells and suggest targeting the LSD1/PLK1 axis as a novel therapeutic approach for lung adenocarcinoma treatment., (©2019 American Association for Cancer Research.)

Caveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT-3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cucurbitacin E (CuE), an active compound of the cucurbitacin family, possesses a variety of pharmacological functions and chemotherapy potential. Cucurbitacin E exhibits inhibitory effects in several types of cancer; however, its anticancer effects on brain cancer remain obscure and require further interpretation. In this study, efforts were initiated to inspect whether CuE can contribute to anti-proliferation in human brain malignant glioma GBM 8401 cells and glioblastoma-astrocytoma U-87-MG cells. An MTT assay measured CuE's inhibitory effect on the growth of glioblastomas (GBMs). A flow cytometry approach was used for the assessment of DNA content and cell cycle analysis. DNA damage 45β (GADD45β) gene expression and CDC2/cyclin-B1 disassociation were investigated by quantitative real-time PCR and Western blot analysis. Based on our results, CuE showed growth-inhibiting effects on GBM 8401 and U-87-MG cells. Moreover, GADD45β caused the accumulation of CuE-treated G2/M-phase cells. The disassociation of the CDC2/cyclin-B1 complex demonstrated the known effects of CuE against GBM 8401 and U-87-MG cancer cells. Additionally, CuE may also exert antitumour activities in established brain cancer cells. In conclusion, CuE inhibited cell proliferation and induced mitosis delay in cancer cells, suggesting its potential applicability as an antitumour agent., (© 2019 The Authors Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Common hallmarks of cancer include the dysregulation of cell cycle progression and the acquisition of genome instability. In tumors, G1 cell cycle checkpoint induction is often lost. This increases the reliance on a functional G2/M checkpoint to prevent progression through mitosis with damaged DNA, avoiding the introduction of potentially aberrant genetic alterations. Treatment of tumors with ionizing radiation (IR) utilizes this dependence on the G2/M checkpoint. Therefore, identification of factors which regulate this process could yield important biomarkers for refining this widely used cancer therapy. Leucine zipper and ICAT domain containing (LZIC) downregulation has been associated with the development of IR-induced tumors. However, despite LZIC being highly conserved, it has no known molecular function. We demonstrate that LZIC knockout (KO) cell lines show a dysregulated G2/M cell cycle checkpoint following IR treatment. In addition, we show that LZIC deficient cells competently activate the G1 and early G2/M checkpoint but fail to maintain the late G2/M checkpoint after IR exposure. Specifically, this defect was found to occur downstream of PIKK signaling. The LZIC KO cells demonstrated severe aneuploidy indicative of genomic instability. In addition, analysis of data from cancer patient databases uncovered a strong correlation between LZIC expression and poor prognosis in several cancers. Our findings suggest that LZIC is functionally involved in cellular response to IR, and its expression level could serve as a biomarker for patient stratification in clinical cancer practice.

Lung cancer is the most common and lethal cancer worldwide, especially in developing countries. Non‑small cell lung cancer (NSCLC) accounts for 85% of all cases of lung cancer. In aprevious study, the protein expression of ubiquitin conjugating enzyme E2 C (UBE2C/UbcH10) in NSCLC tissues and cells was found to be significantly higher than that in adjacent tissues and normal lung epithelial cells. Further study revealed that the aberrant expression of UbcH10 in NSCLC tumors or cancer cells was caused by inactivation of the post‑transcriptional regulation mechanism, and thus microRNAs (miRNAs) may play an important. In the present study, it was demonstrated that the expression of microRNA, hsa‑miR661‑3p, was downregulated and UbcH10 was upregulated in 12 pairs of NSCLC tumors and three NSCLC cell lines. A reporter gene assay revealed that overexpression of hsa‑miR661‑3p effectively reduced the activity of luciferase expressed by a vector bearing the 3' untranslated region of UbcH10 mRNA. Ectopic hsa‑miR661‑3p overexpression mediated by lentiviral infection decreased the expression of UbcH10. Infection of Lv‑miR661‑3p inhibited cell growth and invasion in A549 and SK‑MES‑1 cells. Mechanistically, hsa‑miR661‑3p induced cell cycle G2 arrest through regulation of spindle assembly checkpoint (SAC) function. On the basis of the proposed mechanisms, the objective of the study was to inhibit the proliferation of A549 and SK‑MES‑1 by expressing hsa‑miR661‑3p in vivo and in vitro. Collectively, our results indicated that downregulation of hsa‑miR661‑3p was involved in NSCLC and restoration of hsa‑miR661‑3p impaired the growth of NSCLC cell lines A549 and SK‑MES‑1, suggesting that hsa‑miR661‑3p may be a potential target molecule for the therapy of NSCLC.

The nucleolus is a subnuclear compartment, which governs ribosome biogenesis. Moreover, it functions as hub in the stress response by orchestrating a variety of processes, such as regulation of cell cycle progression, senescence and apoptosis. Emerging evidence links the nucleolus also to the control of genomic stability and the development of human malignancies. Peter Pan (PPAN) is an essential ribosome biogenesis factor localized to nucleoli and mitochondria. We earlier showed that PPAN depletion triggers p53-independent nucleolar stress and apoptosis. In this study we investigated the precise localization of nucleolar PPAN during cell cycle and its function in cell cycle regulation. We show that PPAN knockdown impairs cell proliferation and induces G0/G1 as well as later G2/M cell cycle arrest in cancer cells. Although PPAN knockdown stabilizes the tumor suppressor p53 and induces CDKN1A/p21, the proliferation defects occur largely in a p53/p21-independent manner. We noticed a reduced number of knockdown cells entering cytokinesis and an elevation of binucleation. PPAN knockdown is also associated with increased H2A.X phosphorylation (γH2A.X) in cancer cells. We evaluated a potential signaling axis through the DNA damage response kinases ATM and ATR and alternatively apoptosis as a potent driver of γH2A.X. Interestingly, PPAN knockdown does not involve activation of ATM/ATR. Instead, γH2A.X is generated as a consequence of apoptosis induction in cancer cells. Strikingly, PPAN depletion in human fibroblasts did neither provoke apoptosis nor H2A.X phosphorylation, but recapitulated p53 stabilization. In summary, our data underline the notion that the PPAN-mediated, p53-independent nucleolar stress response has multiple facets., (Copyright © 2019. Published by Elsevier B.V.)

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Maintenance of genome integrity is critical for faithful propagation of genetic information and the prevention of the mutagenesis induced by various DNA damage events. RecQ-mediated genome instability protein 1 (RMI1), together with Bloom syndrome protein and topoisomerase IIIα, form an evolutionarily conserved complex that is critical for the maintenance of genomic stability. Herein, we report that RMI1 depletion increases cell sensitivity to camptothecin treatment, as shown by an elevation of genotoxic stress-induced DNA double-strand breaks, a stronger activation of the DNA damage response, and a greater G2/M cell cycle delay. Our findings support that, upon DNA damage, RMI1 forms nuclear foci at the damaged regions, interacts with RAD51, and facilitates the recruitment of RAD51 to initiate homologous recombination. Our data reveal the importance of RMI1 in response to DNA double-strand breaks and shed light on the molecular mechanisms by which RMI1 contributes to maintain genome stability.-Fang, L., Sun, X., Wang, Y., Du, L., Ji, K., Wang, J., He, N., Liu, Y., Wang, Q., Zhai, H., Hao, J., Xu, C., Liu, Q. RMI1 contributes to DNA repair and to the tolerance to camptothecin.

Altered or aberrant expression of several splicing factors leads to the progression of different cancers. Though there are several ongoing studies underscoring the role of the splicing regulator polypyrimidine tract binding protein 2 (PTBP2) in neuronal cells, we unveil the role of PTBP2 in chronic myeloid leukemia (CML). Different RNA binding proteins (RBP's) earlier reported in chronic myeloid leukemia blast crisis (CML-BC) cases (n = 28) from Radich Oncomine leukemia dataset, were compared. We observed increased expression of MSI2 followed by PTBP2 in BC cases and increased PTBP2 expression in relapsed cases (n = 10) from the same dataset compared to other RBPs. We also observed increased PTBP2 exon 10 inclusion in KCL22, a granulocytic lineage CML cell line when compared to other CML cell lines of different lineages. As PTBP2 protein expression is associated with PTBP2 exon 10 inclusion, we observed in cell lines and in a set of progressed cases (n = 4) that increased BCR-ABL1 expression potentiates PTBP2 exon 10 inclusion and thus confers the existence of a functional protein. Inhibition of BCR-ABL1 with imatinib not only blocks the inclusion of exon 10 but also deregulates PTBP2 expression in CML cells. Knockdown of PTBP2 in KCL22 cells leads to reduced cell proliferation, increased G2/M cell cycle arrest and increased apoptosis. Taken together our study portrays PTBP2 as a new possible target for CML and progressive inclusion/exclusion of PTBP2 exon 10 might play an important role in CML progression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Fusion of critically short or damaged telomeres is associated with the genomic rearrangements that support malignant transformation. We have demonstrated the fundamental contribution of DNA ligase 4-dependent classical non-homologous end-joining to long-range inter-chromosomal telomere fusions. In contrast, localized genomic recombinations initiated by sister chromatid fusion are predominantly mediated by alternative non-homologous end-joining activity that may employ either DNA ligase 3 or DNA ligase 1. In this study, we sought to discriminate the relative involvement of these ligases in sister chromatid telomere fusion through a precise genetic dissociation of functional activity. We have resolved an essential and non-redundant role for DNA ligase 1 in the fusion of sister chromatids bearing targeted double strand DNA breaks that is entirely uncoupled from its requisite engagement in DNA replication. Importantly, this fusogenic repair occurs in cells fully proficient for non-homologous end-joining and is not compensated by DNA ligases 3 or 4. The dual functions of DNA ligase 1 in replication and non-homologous end-joining uniquely position and capacitate this ligase for DNA repair at stalled replication forks, facilitating mitotic progression., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

G2/M-arrested proximal tubular epithelial cells (TECs) after renal injury are linked to increased cytokines production. ATG5-mediated autophagy in proximal TECs has recently been shown to protect against G2/M cell cycle arrest and renal fibrosis. However, the impacts of autophagy in regulating inflammatorily response mounted by injured TECs remains largely unknown. In the present study, we investigated whether ATG5 acts as an innate immune suppressor in proximal TECs during kidney injury. Using the unilateral ureteric obstruction model in proximal tubule-specific autophagy-deficient mice, we demonstrated that ablation of epithelial ATG5 genes markedly impaired autophagy, resulting in enhanced nuclear factor κB (NF-κB) activation, macrophage and lymphocyte infiltration, and proinflammatory cytokines production in obstructed kidneys, as compared with wild-type mice. Following stimulation with angiotensin II (Ang II), siRNA silencing of ATG5 in cultured HK-2 cells or ATG5-deficient primary proximal TECs produced more cytokines, including IL-1β, IL-6, and TNF-α than did their control cells. Overexpressed ATG5, but not the autophagy-incompetent ATG5 mutant K130R in HK-2 cells, rendered resistant to Ang II-induced inflammatory response. Immunofluorescence assay indicated that ATG5 and p65 colocalized in the nucleus and cytoplasm, and their interaction was verified in immunoprecipitation assay from HEK-293T cell extracts. Genetic downregulation of endogenous ATG5 increased Ang II-induced phosphorylation and nuclear translocation of p65 and transcriptional activity of NF-κB, whereas the overexpressed ATG5, rather than ATG5 mutant K130R, hampered activation of NF-κB signaling, suggest an autophagy-dependent anti-inflammatory effect of ATG5. Further, pharmacological manipulation of autophagy yielded similar results both in vivo and in vitro. Additionally, JSH-23, a specific inhibitor of NF-κB nuclear translocation, rescued Ang II-driven IL-1β production in ATG5 siRNA-treated cells and decreased the proportion of cells in G2/M phase. In conclusion, ATG5-mediated autophagy in tubules targets NF-κB signaling to protect against renal inflammation.

Purpose: Totarol is a plant-derived natural product and has been reported to exhibit important pharmacological activities. However, the anticancer activity of totarol has not been evaluated yet. Therefore, the present research work was designed to evaluate the antitumor effects of totarol in SGC-7901 human gastric cancer cells and human gastric epithelial mucosa cell line GES-1 (used as normal cell line model) together with examining its effects on induction of apoptosis, cell cycle phase distribution and cell migration., Methods: The effect of totarol on cell cytotoxicity was evaluated by MTT cell viability assay. Inverted phase contrast microscopy was used to identify the effects on cell morphology, while transmission electron microscopy indicated the apoptosis-driven morphological changes in cancer cells. The effects on cell apoptosis were also evaluated by annexin V/PI staining, while cell cycle analysis was done by flow cytometry. In vitro wound healing assay estimated the effects of totarol on cell migration., Results: The results indicated that totarol induced selective cytotoxic effects in SGC-7901 human gastric cancer cells concentration-dependently and exhibited lower toxicity in GES-1 normal cells. The totarol-treated cells showed significant alterations in cell morphology including rounding and cellular shrinkage. Untreated SGC-7901 cells exhibited normal cellular morphology with undamaged plasma membrane. However, treating cells with totarol led to damaged plasma membrane along with appearance of rounded protrusions (apoptotic bodies) containing damaged and broken chromatin material. Treatment with different doses of totarol led to profound suppression of wound healing. Totarol treatment also led to G2/M phase cell cycle arrest in these cells in a concentration-dependent manner., Conclusions: The present study indicated that totarol diterpene has the tendency to show selective anticancer effects in SGC-7901 human gastric cancer cells along with inducing apoptosis, cell cycle arrest and inhibition of cell migration.

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation., Competing Interests: AC, JJ, GC, IG, MU, AC No competing interests declared, (© 2019, Cosolo et al.)

Aberrant increase in mitochondrial biogenesis is common in human ovarian cancer and has great therapeutic value. In this work, we demonstrate that tigecycline, a FDA-approved broad spectrum antibiotic, selectively targets ovarian cancer cells through inhibition of mitochondrial translation. Tigecycline dose-dependently inhibits proliferation of ovarian cancer cells via arresting them at G2/M phase and induces apoptosis through caspase pathway. At the same concentration, tigecycline either does not or inhibits normal cells in a less extent than ovarian cancer cells. Mechanistically, tigecycline specifically inhibits translation by mitochondrial ribosome but not nuclear or cytosolic ribosome, leading to mitochondrial dysfunction, oxidative stress and damage, AMPK activation and inhibition of mTOR signaling in ovarian cancer cells. We further show that the inhibitory effects on ovarian cancer cell by tigecycline is mediated by its suppression of mitochondrial respiration. Importantly, the combination of tigecycline and cisplatin at sublethal concentration results in much greater efficacy than cisplatin alone in vitro and in vivo. Additionally, the effective dose of tigecycline in ovarian cancer is clinically achievable. Our study suggests that tigecycline is a useful addition to the treatment of ovarian cancer. Our work also highlights the targeted therapeutic potential of mitochondrial respiration in ovarian cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cell cycle is under circadian regulation. Oncogenes can dysregulate circadian-regulated genes to disrupt the cell cycle, promoting tumor cell proliferation. As a regulator of G2/M arrest in response to DNA damage, the circadian gene Timeless Circadian Clock (TIMELESS) coordinates this connection and is a potential locus for oncogenic manipulation. TIMELESS expression was evaluated using RNASeq data from TCGA and by RT-qPCR and western blot analysis in a panel of colon cancer cell lines. TIMELESS expression following ERK inhibition was examined via western blot. Cell metabolic capacity, propidium iodide, and CFSE staining were used to evaluate the effect of TIMELESS depletion on colon cancer cell survival and proliferation. Cell metabolic capacity following TIMELESS depletion in combination with Wee1 or CHK1 inhibition was assessed. TIMELESS is overexpressed in cancer and required for increased cancer cell proliferation. ERK activation promotes TIMELESS expression. TIMELESS depletion increases γH2AX, a marker of DNA damage, and triggers G2/M arrest via increased CHK1 and CDK1 phosphorylation. TIMELESS depletion in combination with Wee1 or CHK1 inhibition causes an additive decrease in cancer cell metabolic capacity with limited effects in non-transformed human colon epithelial cells. The data show that ERK activation contributes to the overexpression of TIMELESS in cancer. Depletion of TIMELESS increases γH2AX and causes G2/M arrest, limiting cell proliferation. These results demonstrate a role for TIMELESS in cancer and encourage further examination of the link between circadian rhythm dysregulation and cancer cell proliferation., Competing Interests: The authors have declared that no competing interests exist.

Background: Accumulating studies discloses that long non-coding RNAs (lncRNAs) serve important roles in human tumorigenesis, including nasopharyngeal carcinoma (NPC). The purpose of the present study was to determine the role of lncRNA FEZF1-AS1 in NPC., Materials and Methods: The expression levels of FEZF1-AS1 in NPC tissues and cell lines were detected by RT-qPCR analysis. MTT assay was performed to investigate the proliferation of NPC cells in vitro , whereas the migration and invasion of NPC cells were determined by wound healing assay and transwell assay. A nude mouse tumor model was established to study the role of FEZF1-AS1 in NPC tumorigenesis in vivo The expression levels of proteins were detected by Western blot assay., Results: The results showed that FEZF1-AS1 expression was increased in the NPC tissues and cell lines, and the higher expression of FEZF1-AS1 was closely associated with poor prognosis of NPC patients. We further observed that knockdown of FEZF1-AS1 inhibited the proliferation of NPC cells in vitro and suppressed NPC xenograft growth in vivo through inducing G2/M cell cycle arrest. The migratory and invasive abilities of NPC cells were also reduced upon FEZF1-AS1 knockdown. Moreover, we demonstrated that inhibition of FEZF1-AS1 remarkably suppressed epithelial-mesenchymal transition (EMT) and reduced β-catenin accumulation in nucleus in NPC cells., Conclusions: Collectively, we showed that FEZF1-AS1 might be a key regulator of cell cycle, EMT and Wnt/β-catenin signaling in NPC cells, which may be helpful for understanding of pathogenesis of NPC., (© 2018 The Author(s).)

The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G 2 /M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca 2 + production and decreased the levels of Δ Ψ m . Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1 α , IRE-1 β , caspase-4, ATF-6 α , Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.

We previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T3 3C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA Here, we show that the RNA-binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Glioblastoma (GBM) is the most common malignant brain tumor and is associated with a poor prognosis, with most patients living less than a year after diagnosis. Given that GBM nearly always recurs after conventional treatments, there is an urgent need to identify novel molecular targets. Hairless (HR) is a nuclear factor enriched in the skin and has been previously implicated in hair cycling. HR is also highly expressed in the brain, but its significance is unknown. We found that human hairless gene (HR) expression is significantly decreased in all GBM subtypes compared with normal brain tissue and is predictive of prognosis, which suggests that loss of HR expression can contribute to GBM pathogenesis. HR was recently discovered to bind to and regulate p53 responsive elements, and thus we hypothesized that HR may have a tumor suppressive function in GBM by modulating p53 target gene expression. We found that HR indeed regulates p53 target genes, including those implicated in cell cycle progression and apoptosis in the GBM-derived U87 cell line, and restoring HR expression triggered G2/M arrest and apoptosis. An analysis of sequenced genomes from patients with GBM revealed 10 HR somatic mutations in patients with glioma, two of which are located in the histone demethylase domain of HR. These two mutations, P996S and K1004N, were reconstructed and found to have impaired p53 transactivating properties. Collectively, the results of our study suggest that HR has tumor suppressive functions in GBM, which may be clinically relevant and a potential avenue for therapeutic intervention., (© 2018 Wiley Periodicals, Inc.)

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.

Gastric cancer, like most of other cancers, has an uncontrolled cell cycle regulated by cyclins and cyclin-dependent kinases (CDKs). In this study, we reported that gastric cancer cells showed an accelerated G2/M transition promoted by CREPT/RPRD1B and Aurora kinase B (Aurora B). We found that CREPT/RPRD1B and Aurora B were coordinately expressed during the cell cycle in gastric cancer cells. Deletion of CREPT/RPRD1B disturbed the cell progression and extended the length of cell cycle, leading to a significant accumulation of mitotic cells. Mechanistically, we revealed that CREPT/RPRD1B interacted with Aurora B to regulate the expression of Cyclin B1 in gastric cancer cells. Interestingly, Aurora B phosphorylates S145 in a well-conserved motif of CREPT/RPRD1B. We proposed that phosphorylation of CREPT/RPRD1B by Aurora B is required for promoting the transcription of Cyclin B1, which is critical for the regulation of gastric tumorigenesis. Our study provides a mechanism by which gastric tumor cells maintain their high proliferation rate via coordination of Aurora B and CREPT/RPRD1B on the expression of Cyclin B1. Targeting the interaction of Aurora B and CREPT/RPRD1B might be a strategy for anti-gastric cancer therapy in the future.

Poor prognoses remain the most challenging aspect of hepatocellular carcinoma (HCC) therapy. Consequently, alternative therapeutics are essential to control HCC. This study investigated the anticancer effects of safranal against HCC using in vitro, in silico, and network analyses. Cell cycle and immunoblot analyses of key regulators of cell cycle, DNA damage repair and apoptosis demonstrated unique safranal-mediated cell cycle arrest at G2/M phase at 6 and 12 h, and at S-phase at 24 h, and a pronounced effect on DNA damage machinery. Safranal also showed pro-apoptotic effect through activation of both intrinsic and extrinsic initiator caspases; indicating ER stress-mediated apoptosis. Gene set enrichment analysis provided consistent findings where UPR is among the top terms of up-regulated genes in response to safranal treatment. Thus, proteins involved in ER stress were regulated through safranal treatment to induce UPR in HepG2 cells.

Proper DNA double-strand break (DSB) repair is essential for maintaining genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism, which introduces mutations at break sites and contributes to chromosomal translocations and telomere fusions, thus driving carcinogenesis. Mitotic kinases PLK1, CDK1 and Aurora A are important for supporting MMEJ and are often overexpressed in various tumors. However, the functional interplay between these kinases and MMEJ has not been explored. Here, we found that MMEJ is preferentially employed to fix DSBs in cells arrested in mitosis following nocodazole treatment. We further showed that the DSB repair factor CtIP is jointly phosphorylated by CDK1/Aurora A and PLK1. CDK1/Aurora A-mediated CtIP phosphorylation at serine 327 triggers CtIP binding to the PLK1 polo-box domain, which in turn facilitates PLK1 to phosphorylate CtIP mainly at serine 723. A PLK1 phosphor-mimic CtIP mutant fails to initiate extended end resection and is thus unable to mediate homologous recombination and the G2/M checkpoint but can mediate MMEJ. These data imply that PLK1 may target CtIP to promote error-prone MMEJ and inactivate the G2/M checkpoint. These findings have helped elucidate the oncogenic roles of these factors.

Malignant melanoma is one of the most fatal and aggressive skin cancers, originating from pigment-containing melanocytes. Despite progress in clinical research, treatment options for malignant melanoma have been limited. The nuclear factor of activated T-cell 5 (NFAT5), originally identified as tonicity regulated transcription factor Ton/EBP, is now known as a carcinogenic gene in several types of cancer pathology. In this study, we knocked down NFAT5 to investigate its role in melanoma cancer. shRNA-mediated knockdown of NFAT5 led to a significant decrease in cell proliferation in vitro. Additionally, depletion of NFAT5 inhibited the cell migratory ability of B16BL6 melanoma cells and led to more accumulation at the G2/M phase of the cell cycle. Furthermore, NFAT5 was essential for the development of melanoma cancer pathophysiology in an in vivo mouse model. NFAT5 knockdown-induced tumor growth was slow and tumor volume was significantly reduced compared to mock controls. Moreover, NFAT5 knockdown was associated with a low number of metastatic nodules on the lung and liver. To our knowledge, our data demonstrate for the first time a role of NFAT5 in the development of melanoma. We provide evidence for NFAT5 as a marker of cell migration and metastasis, indicating that NFAT5 represents a novel therapeutic target in melanoma., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia, are characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Reports on analysis of bone marrow samples from patients have revealed a high incidence of mutations in splicing factors in early stem and progenitor cell clones, but the mechanisms underlying transformation of HSPCs harboring these mutations remain unknown. Using ex vivo cultures of primary human CD34 + cells as a model, we find that mutations in splicing factors SRSF2 and U2AF1 exert distinct effects on proliferation and differentiation of HSPCs. SRSF2 mutations cause a dramatic inhibition of proliferation via a G2-M phase arrest and induction of apoptosis. U2AF1 mutations, conversely, do not significantly affect proliferation. Mutations in both SRSF2 and U2AF1 cause abnormal differentiation by skewing granulo-monocytic differentiation toward monocytes but elicit diverse effects on megakaryo-erythroid differentiation. The SRSF2 mutations skew differentiation toward megakaryocytes whereas U2AF1 mutations cause an increase in the erythroid cell populations. These distinct functional consequences indicate that SRSF2 and U2AF1 mutations have cell context-specific effects and that the generation of myeloid disease phenotype by mutations in the genes coding these two proteins likely involves different intracellular mechanisms. Stem Cells 2018;36:1663-1675., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2018.)

Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), has attracted the interest of hematologists because more than 90% of acute myeloid leukemia (AML) blasts express its target, CD33. Although GO and subsequently developed ADCs depend on lysosomes for activation, lysosome number and activity in tumor cells has not been well elucidated. In this study, we investigated whether an mTORC1/2 kinase inhibitor, PP242, which was reported to activate lysosomal function, potentiates the cytotoxicity of GO in AML cells. Eight AML cell lines (U937, THP-1, SKM-1, SKK-1, SKNO-1, HL-60, MARIMO and KO52) were treated with GO and PP242. The cytotoxic effect of GO was enhanced by concurrent treatment with a non-cytotoxic concentration (500 nM) of PP242 in most cell lines, except MARIMO and KO52 cells. We then used LysoTracker to label acidic lysosomes in U937, THP-1, SKM-1, MARIMO and KO52 cells. LysoTracker fluorescence was dramatically increased by treatment with PP242 in U937, THP-1 and SKM-1 cells, and the intensified fluorescence was retained with PP242 + GO. In contrast, PP242 did not induce a significant increase in fluorescence in MARIMO cells, consistent with the lack of combinatory cytotoxicity. LysoTracker fluorescence was also increased by PP242 in KO52 cells, which have been reported to strongly express multidrug resistance (MDR). Further, PP242 suppressed GO-induced Chk1 activation and G2/M cell cycle arrest, which in turn triggered cell cycle promotion and cell death. These results indicate that inhibition of mTORC1/2 kinase by PP242 enhanced the cytotoxicity of GO by increasing lysosomal compartments and promoting the cell cycle via suppression of GO-induced Chk1 activation. This combination may represent an attractive new therapeutic strategy for the treatment of leukemia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The prognosis of small cell lung cancer (SCLC) is poor despite its good initial response to chemotherapy. Polo-like kinase 1 (PLK1) is a crucial mitotic regulator that is overexpressed in many tumors, and its overexpression is associated with tumor aggressiveness and a poor prognosis. However, its role in SCLC is still poorly characterized. Based on immunohistochemistry findings, the PLK1 protein is expressed at higher levels in SCLC tumor samples than in normal lung tissue samples. The selective PLK1 inhibitor BI 6727 significantly induced the inhibition of proliferation and apoptosis in a dose-dependent manner in SCLC cell lines. FACS analysis showed an increase in the population of cells in the G2/M phase, followed by DNA damage and the consequent activation of the ataxia telangiectasia and Rad3-related (ATR)/ataxia telangiectasia mutated (ATM)-Chk1/Chk2 checkpoint pathway. In addition, BI 6727 treatment resulted in clearly attenuated growth and apoptosis in NCI-H446 xenografts. The level of histone H2AX phosphorylation at serine-139 (γH2AX) was markedly increased both in vitro and in vivo. Our findings indicate that BI 6727 has therapeutic potential for SCLC patients., (Copyright © 2018. Published by Elsevier B.V.)

Background/aim: Nasopharyngeal cancer (NPC) has a high local recurrence rate due to its resistance to ionizing radiation (IR). Replication protein A1 (RPA1) is one of the main elements in the homologous repair (HR) pathway, which is closely associated with the repair of DNA double strand breaks (DDBs). Studies on the relationship between RPA1 and the radiosensitivity of NPC are substantially limited. It was hypothesized that RPA1 plays a crucial role in predicting the radiosensitivity of NPC., Methods: The protein expression of RPA1 in 182 patients with NPC in the complete response (CR) and non-complete response (nCR) groups was evaluated using immunohistochemistry. Then, univariate and multivariate analysis were performed using SPSS software vision 22 to determine the relationship between the expression of RPA1 and the clinicopathological features. In addition, the mRNA expression of RPA1 was tested in 24 fresh samples using qRT-PCR. RPA1 was silenced in CNE-2R cell lines combined with IR to measure the radiosensitivity, proliferation, DNA damage repair and cell cycle of CNE-2R cells. Xenograft models in nude mice were used to determine the effect of RPA1 on tumor growth after IR. Immunoblotting and immunofluorescence staining were performed to identify proteins that interacted with RPA1. All statistical tests were two-sided., Results: RPA1 protein was overexpressed in NPC patients with nCR (65.31%), and was an independent predictor of radiosensitivity (HR: 3.755, 95% CI: 1.990-7.085), in addition to Epstein-Barr virus (EBV; HR: 3.984; 95% CI: 1.524-10.410). The silencing of RPA1 increased the radiosensitivity of CNE-2R cells, blocked the repair of DNA, impaired cell proliferation, and contributed to G2/M cell cycle arrest. Furthermore, the xenograft models in nude mice revealed that silencing RPA1 combined with irradiation significantly retarded the growth of tumors. Moreover, the knockdown of RPA1 decreased Rad51 collection to the damage site and prolonged the time of DNA repair., Conclusion: RPA1 protein is frequently overexpressed in NPC patients with nCR. The silencing of RPA1 enhanced the radiosensitivity of CNE-2R cells. These present findings reveal that RPA1 is a potential biomarker for predicting the radiosensitivity in NPC., (Copyright © 2018. Published by Elsevier Inc.)

This study was designed to research the influence of activating transcription factor 3 (ATF3) on the radioresistance of breast cancer. ATF3 expression was measured by qRT-PCR and immunohistochemistry. Cancerous cell lines were cultured in vitro, and the expression of ATF3 was gauged by both qRT-PCR and Western blot before and after the radiation therapy. Cellular cycle and apoptosis were analysed by flow cytometry. Changes in the expression of corresponding proteins in the downstream pathways were identified by Western blot. Tumour xenograft was used to evaluate the effect of ATF3 in vivo. ATF3 was observed stronger in breast cancer tissues and cells. After radiation therapy, the expression of ATF3 in breast cancer cells was up-regulated. Silencing ATF3 could promote G2/M phase block, facilitate cell apoptosis and decrease clonogenic survival rate. The overexpression of ATF3 could curb G2/M-phase block, cell apoptosis and increase clonogenic survival rate. Overexpression ATF3 could increase radioresistance by up-regulating the level of phosphorylation of Akt in the PI3K/Akt signalling pathway. Radioresistance of breast cancer cells could be alleviated by inhibiting the PI3K/Akt signalling pathway. ATF3 could also promote radioresistance in vivo. ATF3 gene was able to promote radioresistance of breast cancer cells., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G 2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4 + T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G 2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Checkpoint recovery, the process that checkpoint-arrested cells with normal DNA repair capacity resume cell cycle progression, is essential for genome stability. However, the signaling network of the process has not been clearly defined. Here, we combine functional proteomics, mathematical modeling, and molecular biology to identify mTORC1, the nutrient signaling integrator, as the determinant for G2/M checkpoint recovery. Inhibition of the mTORC1 pathway delays mitotic entry after DNA damage through KDM4B-mediated regulation of CCNB1 and PLK1 transcription. Cells with hyper-mTORC1 activity caused by TSC2 depletion exhibit accelerated G2/M checkpoint recovery. Those Tsc2-null cells are sensitive to WEE1 inhibition in vitro and in vivo by driving unscheduled mitotic entry and inducing mitotic catastrophe. These results reveal that mTORC1 functions as a mediator between nutrition availability sensing and cell fate determination after DNA damage, suggesting that checkpoint inhibitors may be used to treat mTORC1-hyperactivated tumors such as those associated with tuberous sclerosis complex.

Double-stranded DNA breaks activate a DNA damage checkpoint in G2 phase to trigger a cell cycle arrest, which can be reversed to allow for recovery. However, damaged G2 cells can also permanently exit the cell cycle, going into senescence or apoptosis, raising the question how an individual cell decides whether to recover or withdraw from the cell cycle. Here we find that the decision to withdraw from the cell cycle in G2 is critically dependent on the progression of DNA repair. We show that delayed processing of double strand breaks through HR-mediated repair results in high levels of resected DNA and enhanced ATR-dependent signalling, allowing p21 to rise to levels at which it drives cell cycle exit. These data imply that cells have the capacity to discriminate breaks that can be repaired from breaks that are difficult to repair at a time when repair is still ongoing.

The Vestigial-Like Family Member 4 (VGLL4) functions as a native inhibitor of cell proliferation and tumor growth through multiple signaling pathways. We first discovered that VGLL4 causes G2/M phase arrest in hepatocellular carcinoma (HCC) cells. Then, we designed a novel survivin-regulated oncolytic adenovirus Ad-sp-VGLL4 carrying the VGLL4 gene. Ad-sp-VGLL4 exerted high HCC-targeting-selectivity but is less harmful to normal cells. This adenovirus construction enhanced antitumor activity due to G2/M phase arrest and enhanced apoptosis. It's also indicated that Ad-sp-VGLL4 could suppress the growth of transplanted tumor of HCC in vivo experiment. Taken together, our results suggest that Ad-sp-VGLL4 possesses strong antitumor capacity and has great potential use for HCC therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Emerging evidence has indicated the important function of long non-coding RNAs (lncRNAs) in tumour chemotherapy resistance. However, the underlying mechanism is still ambiguous. In this study, we investigate the physiopathologic role of lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) on the paclitaxel (PTX) resistance in breast cancer. Results showed that lncRNA FTH1P3 was up-regulated in paclitaxel-resistant breast cancer tissue and cells (MCF-7/PTX and MDA-MB-231/PTX cells) compared with paclitaxel-sensitive tissue and parental cell lines (MCF-7, MDA-MB-231). Gain- and loss-of-function experiments revealed that FTH1P3 silencing decreased the 50% inhibitory concentration (IC50) value of paclitaxel and induced cell cycle arrest at G2/M phase, while FTH1P3-enhanced expression exerted the opposite effects. In vivo, xenograft mice assay showed that FTH1P3 silencing suppressed the tumour growth of paclitaxel-resistant breast cancer cells and ABCB1 protein expression. Bioinformatics tools and luciferase reporter assay validated that FTH1P3 promoted ABCB1 protein expression through targeting miR-206, acting as a miRNA "sponge." In summary, our results reveal the potential regulatory mechanism of FTH1P3 on breast cancer paclitaxel resistance through miR-206/ABCB1, providing a novel insight for the breast cancer chemoresistance., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Growing evidence indicates a potential role for miR‑490‑3p in tumorigenesis. However, its function in colorectal carcinoma (CRC) remains undefined. In this study, miR‑490‑3p was markedly downregulated in fifty colorectal cancer tissue samples compared with the corresponding adjacent non‑cancerous specimens, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of miR‑490‑3p were closely associated with tumor differentiation and distant metastasis. In addition, both Kaplan-Meier and multivariate analyses indicated CRC patients with elevated miR‑490‑3p amounts had prolonged overall survival. Overexpression of miR‑490‑3p markedly reduced proliferation, colony formation and invasion in CRC cells by enhancing apoptosis and promoting G2/M phase arrest. Furthermore, ectopic expression of miR‑490‑3p resulted in decreased expression of RAB14, which was directly targeted by miR‑490‑3p, as shown by the dual-luciferase reporter gene assay. Finally, in a nude mouse model, miR‑490‑3p overexpression significantly suppressed the growth of CRC cells. The above results indicated that miR‑490‑3p might constitute a prognostic indicator and a novel molecular target for miRNA-based CRC therapy.

Radiation therapy is an important treatment modality for esophageal cancer. However, acquisition of radioresistance ultimately results in esophageal cancer relapse. CD59, a membrane-bound complement regulatory protein, can transduce signals via a Src kinase in the lipid raft, thus playing a complement-independent role. However, the effect of CD59 on the esophageal cancer response to ionizing radiation remains unclear. In this study, we found that the expression level of CD59 was positively correlated with the radioresistance of esophageal cancer cell lines and clinical specimens. High CD59 expression indicated poor overall survival (OS) and disease-free survival (DFS) in esophageal squamous cell carcinoma (ESCC) patients who received radiotherapy. Genetic alteration of CD59 expression modulated the radiosensitivity of esophageal cancer cells to ionizing radiation. CD59 deficiency exacerbated DNA damage, hindered cell proliferation, and induced G2/M cell cycle arrest and cellular senescence, leading to an impaired DNA damage repair ability. In addition, CD59 deficiency almost completely reduced the phosphorylation of Src at Y416 despite ionizing radiation. A Src inhibitor saracatinib sensitized esophageal cancer cells to irradiation. Therefore, CD59 may be a potential biomarker for predicting the radioresistance of ESCC to radiotherapy.