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Haematococcus pluvialis has been used to produce the ketocarotenoid antioxidant, astaxanthin. Currently, heterotrophic cultivation of H. pluvialis is limited by slow growth rates. This work aimed to address this challenge by exploring the mechanisms of acetate metabolism in Haematococcus. Chemical mutagenesis and screening identified H. pluvialis strain KREMS 23D‐3 that achieved up to a 34.9% higher cell density than the wild type when grown heterotrophically on acetate. An integrative proteomics and phosphoproteomics approach was employed to quantify 4955 proteins and 5099 phosphorylation sites from 2505 phosphoproteins in the wild‐type and mutant strains of H. pluvialis. Among them, 12 proteins were significantly upregulated and 22 significantly downregulated in the mutant while phosphoproteomic analysis identified 143 significantly upregulated phosphorylation sites on 106 proteins and 130 downregulated phosphorylation sites on 114 proteins. Upregulation of anaphase‐promoting complex phosphoproteins and downregulation of a putative cell cycle division 20 phosphoprotein in the mutant suggests rapid mitotic progression, coinciding with higher cell division rates. Upregulated coproporphyrinogen oxidase and phosphorylated magnesium chelatase in the mutant demonstrated altered nitrogen partitioning toward chlorophyll biosynthesis. The large proportion of differentially expressed phosphoproteins suggests phosphorylation is a key regulator for protein expression and activity in Haematococcus. Taken together, this study reveals the regulation of interrelated acetate metabolic pathways in H. pluvialis and provides protein targets that may guide future strain engineering work. [ABSTRACT FROM AUTHOR]

The growth arrest and DNA damage inducible (GADD)45 family includes three small and ubiquitously distributed proteins (GADD45A, GADD45B, and GADD45G) that regulate numerous cellular processes associated with stress signaling and injury response. Here, we provide a comprehensive review of the current literature investigating GADD45A, the first discovered member of the family. We first depict how its levels are regulated by a myriad of genotoxic and non‐genotoxic stressors, and through the combined action of intricate transcriptional, posttranscriptional, and even, posttranslational mechanisms. GADD45A is a recognized tumor suppressor and, for this reason, we next summarize its role in cancer, as well as the different mechanisms by which it regulates cell cycle, DNA repair, and apoptosis. Beyond these most well‐known actions, GADD45A may also influence catabolic and anabolic pathways in the liver, adipose tissue and skeletal muscle, among others. Not surprisingly, GADD45A may trigger AMP‐activated protein kinase activity, a master regulator of metabolism, and is known to act as a transcriptional coregulator of numerous nuclear receptors. GADD45A has also been reported to display a cytoprotective role by regulating inflammation, fibrosis and oxidative stress in several organs and tissues, and is regarded an important contributor for the development of heart failure. Overall data point to that GADD45A may play an important role in metabolic, neurodegenerative and cardiovascular diseases, and also autoimmune‐related disorders. Thus, the potential mechanisms by which dysregulation of GADD45A activity may contribute to the progression of these diseases are also reviewed below. [ABSTRACT FROM AUTHOR]

In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict. [ABSTRACT FROM AUTHOR]

RB regulation of E2F is an important component of a highly conserved cell cycle machine, but the consequences of RB loss are mostly context-specific. ChIP-seq studies indicate that RB does not act exclusively at promoters but is also associated with enhancers and chromatin insulators. Cell cycle transitions alter the distribution of RB; when cells proliferate, RB redistributes away from promoters and toward enhancers. RB-binding sites in promoters are conserved; in contrast, RB-binding sites in nonpromoter regions are largely cell type-specific and are mostly independent of E2F. RB's cell type-specific interactions with enhancers and insulators may help to explain some of RB's context-specific activities and its noncanonical functions. The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's requirement in animal development, is tissue-specific, context-specific, and sometimes appears uncoupled from cell proliferation. Detailed new information about RB's genomic distribution provides a new perspective on the complexity of RB function, suggesting that some of its functional specificity results from context-specific RB association with chromatin. Here we summarize recent evidence showing that RB targets different types of chromatin regulatory elements at different cell cycle stages. RB controls traditional RB/E2F targets prior to S-phase, but, when cells proliferate, RB redistributes to cell type-specific chromatin loci. We discuss the broad implications of the new data for RB research. [ABSTRACT FROM AUTHOR]

BACKGROUND: Distinct endothelial cell cycle states (early G1 versus late G1) provide different “windows of opportunity” to enable the differential expression of genes that regulate venous versus arterial specification, respectively. Endothelial cell cycle control and arteriovenous identities are disrupted in vascular malformations including arteriovenous shunts, the hallmark of hereditary hemorrhagic telangiectasia (HHT). To date, the mechanistic link between endothelial cell cycle regulation and the development of arteriovenous malformations (AVMs) in HHT is not known. METHODS: We used BMP (bone morphogenetic protein) 9/10 blocking antibodies and endothelial-specific deletion of activin A receptor like type 1 (Alk1) to induce HHT in Fucci (fluorescent ubiquitination-based cell cycle indicator) 2 mice to assess endothelial cell cycle states in AVMs. We also assessed the therapeutic potential of inducing endothelial cell cycle G1 state in HHT to prevent AVMs by repurposing the Food and Drug Administration–approved CDK (cyclin-dependent kinase) 4/6 inhibitor (CDK4/6i) palbociclib. RESULTS: We found that endothelial cell cycle state and associated gene expressions are dysregulated during the pathogenesis of vascular malformations in HHT. We also showed that palbociclib treatment prevented AVM development induced by BMP9/10 inhibition and Alk1 genetic deletion. Mechanistically, endothelial cell late G1 state induced by palbociclib modulates the expression of genes regulating arteriovenous identity, endothelial cell migration, metabolism, and VEGF-A (vascular endothelial growth factor A) and BMP9 signaling that collectively contribute to the prevention of vascular malformations. CONCLUSIONS: This study provides new insights into molecular mechanisms leading to HHT by defining how endothelial cell cycle is dysregulated in AVMs because of BMP9/10 and Alk1 signaling deficiencies, and how restoration of endothelial cell cycle control may be used to treat AVMs in patients with HHT. [ABSTRACT FROM AUTHOR]

Meiosis is a specialized cell division program that is essential for sexual reproduction. The two meiotic divisions reduce chromosome number by half, typically generating haploid genomes that are packaged into gametes. To achieve this ploidy reduction, meiosis relies on highly unusual chromosomal processes including the pairing of homologous chromosomes, assembly of the synaptonemal complex, programmed formation of DNA breaks followed by their processing into crossovers, and the segregation of homologous chromosomes during the first meiotic division. These processes are embedded in a carefully orchestrated cell differentiation program with multiple interdependencies between DNA metabolism, chromosome morphogenesis, and waves of gene expression that together ensure the correct number of chromosomes is delivered to the next generation. Studies in the budding yeast Saccharomyces cerevisiae have established essentially all fundamental paradigms of meiosis-specific chromosome metabolism and have uncovered components and molecular mechanisms that underlie these conserved processes. Here, we provide an overview of all stages of meiosis in this key model system and highlight how basic mechanisms of genome stability, chromosome architecture, and cell cycle control have been adapted to achieve the unique outcome of meiosis. [ABSTRACT FROM AUTHOR]

Soft tissue and bone sarcomas are a heterogeneous group of malignancies, each with a unique biology and clinical course. As our understanding of individual sarcoma subtypes and their molecular landscapes increases, predictive biomarkers are emerging to improve patient selection for chemotherapies, targeted therapies, and immunotherapy approaches. This review highlights predictive biomarkers rooted in molecular mechanisms of sarcoma biology, focusing on cell cycle regulation, DNA damage repair, and immune microenvironment interactions. We review CDK4/6 inhibitor predictive biomarkers, including CDKN2A loss, ATRX status, MDM2 levels, and Rb1 status. We discuss homologous recombination deficiency (HRD) biomarkers that predict vulnerability to DNA damage repair (DDR) pathway inhibitors, such as molecular signatures and functional HRD markers. We describe tertiary lymphoid structures and suppressive myeloid cells in the sarcoma immune microenvironment that may influence immunotherapy efficacy. While predictive biomarkers are not routinely used in sarcoma clinical practice currently, emerging biomarkers are being developed alongside clinical advancements. Novel therapies and predictive biomarkers will be essential for individualizing future approaches to sarcoma management and improving patient outcomes. [ABSTRACT FROM AUTHOR]

Background: Metastatic breast cancer is associated with a poor prognosis and therefore, innovative therapies are urgently needed. Here, we report on the results of a Phase I-II study using DeltaRex-G for chemotherapy resistant metastatic carcinoma of breast.Patients and Methods:Endpoints: Dose limiting toxicity; Antitumor activity. Eligibility: ≥18 years of age, pathologic diagnosis of breast carcinoma, adequate hematologic and organ function. Treatment: Dose escalation of DeltaRex-G 1-4 x 1011cfu intravenously thrice weekly x 4 weeks with 2-week rest period. Treatment cycles repeated if there is ≤ Grade 1 toxicity until disease progression or unacceptable toxicity. Safety: NCI CTCAE v3 for adverse events reporting, vector related testing. Efficacy: RECIST v1.0, International PET criteria and Choi criteria for response, progression free and overall survival.Results: Twenty patients received escalating doses of DeltaRex-G from 1 × 1011 cfu to 4 × 1011 cfu thrice weekly for 4 weeks with a 2-week rest period. Safety: ≥ Grade 3 treatment-related adverse event: pruritic rash (n = 1), no dose limiting toxicity, no replication-competent retrovirus, nor vector-neutralizing antibodies detected. No vector DNA integration was observed in peripheral blood lymphocytes evaluated. Efficacy: by RECIST v1.0: 13 stable disease, 4 progressive disease; tumor control rate 76%; by PET and Choi Criteria: 3 partial responses, 11 stable disease, 3 progressive disease; tumor control rate 82%. Combined median progression free survival by RECIST v1.0, 3.0 months; combined median overall survival, 20 months; 1-year overall survival rate 83% for Dose Level IV. Biopsy of residual tumor in a participant showed abundant CD8+ killer T-cells and CD45+ macrophages suggesting an innate immune response. Two patients with pure bone metastases had >12-month progression free survival and overall survival and are alive 12 years from the start of DeltaRex-G therapy. These patients further received DeltaRex-G + DeltaVax for 6 months.Conclusion: Taken together, these data indicate that 1) DeltaRex-G has a distinctively high level of safety and exhibits anti-cancer activity, 2) PET/Choi provide a higher level of sensitivity in detecting early signs of tumor response to DeltaRex-G, 3) DeltaRex-G induced 12- year survival in 2 patients with pure bone metastases who subsequently received DeltaVax immunotherapy, and 4) DeltaRex-G may prove to be a biochemical and/or immune modulator when combined with other cancer therapy/immunotherapy.

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programmes. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. Notochord and muscle cells stop dividing after eight or nine rounds of cell division depending on their lineages. In the present study, we showed that a Cdk inhibitor, Cdkn1.b, is responsible for stopping cell cycle progression in these lineages. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells continue to divide even after hatching, and Myc is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by transcription factors that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm is under control of transcription factors that specify the developmental fate of mesenchyme and endoderm. Thus, cell fate specification and cell cycle control are linked by these transcription factors. [ABSTRACT FROM AUTHOR]

In the budding yeast Saccharomyces cerevisiae , exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cells. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem1 GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem1 GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization to the SPB. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem1 GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios., Competing Interests: Competing interests statement:The authors declare no competing interest.

The distribution of allele frequencies of polymorphic markers of the genes of the apoptosis and cell cycle control system TP53 (rs1042522), MDM2 (rs2279744), and CDKN1A (rs1801270) was obtained. The relationship of these markers with the risk of ovarian cancer was analyzed in women of the Moscow region. The study included 70 healthy female donors and 66 patients with ovarian cancer. DNA isolated from the blood of healthy donors and tissue and blood of patients with ovarian cancer was studied. The polymorphic markers of genes were analyzed by real-time PCR with fluorescent allele-specific probes. In the work, the odds ratios (OR) of ovarian cancer risk were obtained: OR = 0.94, 95% CI = 0.57–1.65, p = 1.0 for the Pro allele of the Arg72Pro of the TP53 gene; OR = 0.96, 95% CI = 0.49–1.89, p = 0.90 for the G allele of the T(–410)G of the MDM2 gene; OR = 1.53, 95% CI = 0.76–3.09, p = 0.29 for the Arg allele of the Ser31Arg of the CDKN1A gene. Obtained results show no association with ovarian cancer for studied markers. It was revealed that the carriage of minor alleles of the Arg72Pro of the TP53 gene, T(–410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene did not affect the risk of development of ovarian cancer in women of the Moscow region. [ABSTRACT FROM AUTHOR]

Soft tissue sarcoma is a rare neoplasm of mesenchymal origin, accounting for only ~1% of all adult cancers and consisting of 75 histological subtypes. In the present report, the unique case of a 14 year-old female with metastatic malignant peripheral nerve sheath tumor (formerly, malignant melanotic schwannoma) of the parotid gland, who experienced a durable response and sustained tumor control with Rexin-G®, a tumor-targeted retroviral expression vector encoding an anti-cyclin G1 construct, is described. Post-parotidectomy, and prior to the administration of Rexin-G®, the patient received various chemotherapy regimens, including doxorubicin, ifosfamide, temozolomide, sorafenib, and an immunological therapy with interleukin-2, which only resulted in the further progression of lung metastases. The patient subsequently participated in a Phase 1/2 gene therapy study, during which she received intravenous Rexin-G® as monotherapy for two years with minimal drug-associated adverse events. Currently, the patient has no evidence of active disease 9 years after commencing the Rexin-G® treatment, and with no additional anti-cancer therapy. In conclusion, Rexin-G® may be a viable therapeutic option for malignant peripheral nerve sheath tumors, and should be further investigated in prospective histology-specific clinical trials for this type, and possibly other types, of chemotherapy-resistant sarcoma.

In eukaryotes, cyclin-dependent kinases (CDKs) are required for the onset of DNA replication and mitosis, and distinct CDK–cyclin complexes are activated sequentially throughout the cell cycle. It is widely thought that specific complexes are required to traverse a point of commitment to the cell cycle in G1, and to promote S-phase and mitosis, respectively. Thus, according to a popular model that has dominated the field for decades, the inherent specificity of distinct CDK–cyclin complexes for different substrates at each phase of the cell cycle generates the correct order and timing of events. However, the results from the knockouts of genes encoding cyclins and CDKs do not support this model. An alternative "quantitative" model, validated by much recent work, suggests that it is the overall level of CDK activity (with the opposing input of phosphatases) that determines the timing and order of S-phase and mitosis. We take this model further by suggesting that the subdivision of the cell cycle into discrete phases (G0, G1, S, G2, and M) is outdated and problematic. Instead, we revive the "continuum" model of the cell cycle and propose that a combination with the quantitative model better defines a conceptual framework for understanding cell cycle control. [ABSTRACT FROM AUTHOR]

BackgroundUnderstanding how organisms respond and adapt to environmental changes is central to evolutionary biology. As a sessile organism that has adapted to life in estuaries and intertidal zones, the eastern oyster Crassostrea virginica can tolerate wide fluctuations in temperature and salinity and survive for weeks out of water. To understand the molecular mechanisms underlying the remarkable stress tolerance of the eastern oyster, we studied the transcriptomic changes induced by exposure to air and cold stress. Eastern oysters were maintained for 7 days under four conditions, namely, in seawater (normal) at 22°C, in air at 22°C, in seawater at 5°C and in air at 5°C, and then sampled for RNA sequencing.ResultsTranscriptomic analysis revealed that many genes involved in cell cycle progression and DNA replication were downregulated in oysters exposed to air and cold, which indicates that stress inhibits cell division. Exposure to air at 22°C induced a concerted inhibition of apoptosis through the upregulation of expanded inhibitors of apoptosis and the downregulation of caspases. Interactions between TNF and NF-κB signalling implied a reduction in the inflammatory response and immune functions. Key genes for proline production, fatty acid synthesis and chromosomal proteins were upregulated during exposure to low temperatures, which suggested that proline accumulation, energy conservation, and epigenetic modification of chromosomes are important for coping with cold stress. The upregulation of melatonin, FMRFamide, and neural acetylcholine receptors indicate the significance of the neurohormonal regulation of homeostasis.ConclusionThese results show that air exposure and cold stress alter the expression of key genes for cell division, apoptosis, proline accumulation, fatty acid metabolism, neurohormonal signalling, and epigenetic modifications, suggesting regulation of these processes plays an important role in the stress response of the eastern oyster and possibly other marine molluscs. This study provides new insights into molecular mechanisms of stress response that are essential for understanding the adaptive potential of marine organisms under climate change.

Objective: As a result of TP53 gene mutation high grade serous ovarian cancer (HGSOC) is dependent on the G2 checkpoint for the repair of DNA damage and survival. The key role of WEE1 kinase at this checkpoint makes inhibition of WEE1 kinase in combination with DNA damaging agents an attractive therapeutic strategy for HGSOC. Our aim was to characterise resistance mechanisms to WEE1 inhibitor AZD1775 and identify ways to overcome resistance ready for use in the clinic. Methods: AZD1775-resistant HGSOC cell clones were isolated and western blotting, cell cycle analysis, growth assays, RNA-Seq and gene expression analysis were used to characterise resistance mechanisms and investigate a way to overcome resistance. Results: A resistance mechanism previously reported in small cell lung cancer did not operate in HGSOC. Instead, resistance resulted from different cell cycle control pathway changes that slow AZD1775-induced cell cycle progression and reduce accumulation of replication associated DNA damage. One major change was reduced levels of CDK1, the substrate for WEE1 kinase inhibition; another was increased levels of PKMYT1, which can also inhibit CDK1. Increased expression of TGFβ signalling to slow cell cycle progression occurred in resistant clones. A TGFβR1 inhibitor overcame resistance in a clone with the highest TGFβR1 receptor expression. Conclusions: Although overexpression of the membrane glycoprotein MDR1 is a common mechanism of drug resistance, it was not involved in our HGSOC cells. Instead AZD1775 resistance resulted from cell cycle control pathway changes that combine to slow AZD1775-induced cell cycle progression and so reduce accumulation of replicationassociated DNA damage. [ABSTRACT FROM AUTHOR]

Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

We analyzed associations of polymorphic markers of DNA repair genes (XRCC1, ERCC2), cell cycle control genes (TP53, MDM2, and CDKN1A), methylation of promoter region, and mutation 5382insC of BRCA1 gene in ovarian cancer with effectiveness of platinumbased chemotherapy assessed by the median of progression-free survival time for markers of DNA repair genes and by relapse risk for all studied markers. An increase in the median of progression-free survival time for carriers of the Gln allele (р=0.025) and Gln/Gln genotype (р=0.022) of the Gln399Arg XRCC1 was observed during the 19-months period after chemotherapy. In carriers of C/C genotype of 5382insC mutation of BRCA1 gene (n=6), no relapses were observed (р=0.035), while 17 of 49 patients without this mutation developed relapses. Of 14 patients with BRCA1 gene function inactivation due to promoter methylation or the presence of the C/C genotype of 5382insC, one relapse was observed (p=0.033). Multivariate analysis revealed an association of markers of the XRCC1, TP53, MDM2 genes, BRCA1 gene inactivation, and type of surgery with the risk of relapse during the follow-up period up to 19 months after the end of chemotherapy (р≤0.0007). [ABSTRACT FROM AUTHOR]

Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the Swi4/Swi6 (SBF) complex that promotes transcription of the cyclins CLN1 and CLN2. In late G1, Whi5 is rapidly hyper-phosphorylated by Cln1 and Cln2 in complex with the cyclin-dependent kinase Cdk1. This hyper-phosphorylation inactivates Whi5 and excludes it from the nucleus. Here, we set out to determine the molecular mechanisms responsible for Whi5's multi-site phosphorylation and how they regulate the cell cycle. To do this, we first identified the 19 Whi5 sites that are appreciably phosphorylated and then determined which of these sites are responsible for G1 hypo-phosphorylation. Mutation of 7 sites removed G1 hypo-phosphorylation, increased cell size, and delayed the G1/S transition. Moreover, the rapidity of Whi5 hyper-phosphorylation in late G1 depends on "priming" sites that dock the Cks1 subunit of Cln1,2-Cdk1 complexes. Hyper-phosphorylation is crucial for Whi5 nuclear export, normal cell size, full expression of SBF target genes, and timely progression through both the G1/S transition and S/G2/M phases. Thus, our work shows how Whi5 phosphorylation regulates the G1/S transition and how it is required for timely progression through S/G2/M phases and not only G1 as previously thought. [Display omitted] • The cell-cycle inhibitor Whi5 is hypo-phosphorylated in G1 on 7 sites • Mutation of the 7 G1 phosphosites increases cell size and delays G1/S • Whi5 hyper-phosphorylation at the G1/S transition depends on Cks1 • Whi5 hyper-phosphorylation promotes transcription and cell-cycle progression In budding yeast, the cell-cycle inhibitor Whi5 is hyper-phosphorylated at the G1/S transition by Cdk1 in complex with its Cks1 subunit. Xiao et al. identify 7 sites that are hypo-phosphorylated in early G1. Disruption of Whi5 phosphorylation before or after Start causes an increase in cell size, cell-cycle delays, and reduced gene expression. [ABSTRACT FROM AUTHOR]

Subversion of genome integrity fuels cellular adaptation and is a prerequisite for organismal evolution, yet genomic lesions are also the harmful driving force of cancer and other age-related human diseases. Genome integrity maintenance is inherently linked to genome organization and nuclear architecture, which are substantially remodeled during the cell cycle. Here we discuss recent findings on how actively dividing cells cope with endogenous genomic lesions that occur frequently at repetitive, heterochromatic, and late replicating regions as byproducts of genome duplication. We discuss how such lesions, rather than being resolved immediately when they occur, are dealt with in subsequent cell cycle phases, and even after mitotic cell division, and how this in turn affects genome organization, stability, and function. [Display omitted] • Repetitive, heterochromatic and late replicating regions are inherently fragile. • Replication stress intermediates take more than one cell cycle to be resolved. • Mitotic DNA synthesis precedes LLPS-mediated lesion compartmentalization in G1. • Genomic lesions segregated over multiple cell generations shape cancer genomes. [ABSTRACT FROM AUTHOR]

Background: Lipopolysaccharide (LPS), a structural and protective compound primarily found in ordinary benign bacteria, could induce pro-inflammatory effect in a macrophage cell line Raw264.7 cells. Additionally, we previously showed that oleic acid (OA) possessed apoptotic effect in mouseoriginated Raw264.7 cells as well. Methods: Cellular and molecular methods including flowcytometry and Western blot as well as bioinformatic methods including RNAseq and analysis. Results: We demonstrated that OA could alleviate LPS-activated inflammatory effects, including apoptosis and secretion of cytokines via the modulation of cell cycle process. Further analysis revealed that OA reduced the LPS-elevated expression of p21, but not p16. Conclusion: Our investigation has provided detailed information on LPS stimulation and OA remission in Raw264.7 cells, and laid solid foundation for the potential pharmaceutical application of OA as an antiinflammatory agent.

Cell cycle checkpoints secure ordered progression from one cell cycle phase to the next. They are important to signal cell stress and DNA lesions and to stop cell cycle progression when severe problems occur. Recent work suggests, however, that the cell cycle control machinery responds in more subtle and sophisticated ways when cells are faced with naturally occurring challenges, such as replication impediments associated with endogenous replication stress. Instead of following a stop and go approach, cells use fine-tuned deceleration and brake release mechanisms under the control of ataxia telangiectasia and Rad3-related protein kinase (ATR) and checkpoint kinase 1 (CHK1) to more flexibly adapt their cell cycle program to changing conditions. We highlight emerging examples of such intrinsic cell cycle checkpoint regulation and discuss their physiological and clinical relevance. Shutdown of cell cycle progression by checkpoint activation is the exception rather than the norm when cells face physiological levels of replication stress. In response to endogenous replication stress, cells use tunable deceleration and brake release mechanisms under the control of the ATR and CHK1 kinases for timely completion of DNA duplication. Intrinsic checkpoint activation, maintenance, and recovery represent a continuum, which is modulated by CHK1 phosphorylation and ubiquitin-dependent proteasomal degradation. Sharp cell cycle transitions, in which one cell cycle phase is fully concluded before the next one begins, can be blurred due to a balancing act between genome integrity maintenance and an urge for cell cycle completion. Deviations from ordered cell cycle phase transitions promote cancer, with therapeutic opportunities for cell cycle checkpoint kinase inhibitors. [ABSTRACT FROM AUTHOR]

Cyclins are key regulators of cell cycle progression and survival. Particularly cyclins D (cyclin D1, D2, and D3) act in response to the mitogenic stimulation and are pivotal mediators between proliferative pathways and the nuclear cell cycle machinery. Dysregulation of cyclins expression results in impaired development, abnormal cell growth or tumorigenesis.In this review we summarize current knowledge about regulatory role of the cyclin D promoters, transcriptional factors: regulators, co-activators and adaptor proteins necessary to their activation. We focused on the intracellular signaling pathways vital to cell growth, differentiation and apoptosis including transcription factor families: activator protein 1 (AP1), nuclear factor (NFκB), signal transducer and activator of transcription (STAT), cAMP response element-binding protein (CREB) and Sp/NF-Y, with a special insight into the tissue specific cyclin representation.

Abstract Background Evolutionarily conserved RFX transcription factors (TFs) regulate their target genes through a DNA sequence motif called the X-box. Thereby they regulate cellular specialization and terminal differentiation. Here, we provide a comprehensive analysis of all the eight human RFX genes (RFX1–8), their spatial and temporal expression profiles, potential upstream regulators and target genes. Results We extracted all known human RFX1–8 gene expression profiles from the FANTOM5 database derived from transcription start site (TSS) activity as captured by Cap Analysis of Gene Expression (CAGE) technology. RFX genes are broadly (RFX1–3, RFX5, RFX7) and specifically (RFX4, RFX6) expressed in different cell types, with high expression in four organ systems: immune system, gastrointestinal tract, reproductive system and nervous system. Tissue type specific expression profiles link defined RFX family members with the target gene batteries they regulate. We experimentally confirmed novel TSS locations and characterized the previously undescribed RFX8 to be lowly expressed. RFX tissue and cell type specificity arises mainly from differences in TSS architecture. RFX transcript isoforms lacking a DNA binding domain (DBD) open up new possibilities for combinatorial target gene regulation. Our results favor a new grouping of the RFX family based on protein domain composition. We uncovered and experimentally confirmed the TFs SP2 and ESR1 as upstream regulators of specific RFX genes. Using TF binding profiles from the JASPAR database, we determined relevant patterns of X-box motif positioning with respect to gene TSS locations of human RFX target genes. Conclusions The wealth of data we provide will serve as the basis for precisely determining the roles RFX TFs play in human development and disease.

Contamination of soil and water with heavy metals and metalloids is a serious environmental problem. Cadmium and arsenic are major environmental contaminants that pose a serious threat to human health. Although toxicities of cadmium and arsenic to living organisms have been extensively studied, the molecular mechanisms of cellular responses to cadmium and arsenic remain poorly understood. In this study, we demonstrate that the cell wall integrity (CWI) pathway is involved in coping with cell wall stresses induced by cadmium and arsenate through its role in the regulation of cell wall modification. Interestingly, the Rlm1p and SBF (Swi4p-Swi6p) complex transcription factors of the CWI pathway were shown to be specifically required for tolerance to cadmium and arsenate, respectively. Furthermore, we found the PIR2 gene, encoding cell wall O-mannosylated heat shock protein, whose expression is under the control of the CWI pathway, is important for maintaining cell wall integrity during cadmium and arsenate stresses. In addition, our results revealed that the CWI pathway is involved in modulating the expression of genes involved in cell wall biosynthesis and cell cycle control in response to cadmium and arsenate via distinct sets of transcriptional regulators. [ABSTRACT FROM AUTHOR]

Meiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes an STE20 family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20 MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15. [ABSTRACT FROM AUTHOR]

We studied the association of polymorphic markers of cell cycle control genes (Arg72Pro of the TP53 gene, T(-410)G of the MDM2 gene, and Ser31Arg of the CDKN1A gene) in ovarian cancer and progression-free survival following platinum-based chemotherapy. Tumor tissue samples obtained from 49 patients who had undergone chemotherapy were examined. Patients received standard platinum-based chemotherapy and were observed until disease progression. Polymorphic markers of genes were evaluated by PCR-RFLP and real-time PCR. In patients carrying the G allele of the T(-410)G marker of the MDM2 gene, a decreasing trend was observed in median progression-free survival. An increase in the median progression-free survival was observed in carriers of the Pro allele of the TP53 gene (p=0.045). Furthermore, a stronger association was noted with carriers of the minor Pro/Pro homozygous genotype relative to the Arg/Arg genotype (p=0.007). In the subgroup of patients who underwent optimal or complete cytoreductive surgery, carriage of the minor Arg allele of the Ser31Arg marker (CDN1A gene) was associated with a decrease in the median progression-free survival time (p=0.004). [ABSTRACT FROM AUTHOR]

Senescent cells may possess the intrinsic programs of metabolic and epigenomic remodeling, but the molecular mechanism remains to be clarified. Using an RNAi‐based screen of chromatin regulators, we found that knockdown of the NSD2/WHSC1/MMSET methyltransferase induced cellular senescence that augmented mitochondrial mass and oxidative phosphorylation in primary human fibroblasts. Transcriptome analysis showed that loss of NSD2 downregulated the expression of cell cycle‐related genes in a retinoblastoma protein (RB)‐mediated manner. Chromatin immunoprecipitation analyses further revealed that NSD2 was enriched at the gene bodies of actively transcribed genes, including cell cycle‐related genes, and that loss of NSD2 decreased the levels of histone H3 lysine 36 trimethylation (H3K36me3) at these gene loci. Consistent with these findings, oncogene‐induced or replicative senescent cells showed reduced NSD2 expression together with lower H3K36me3 levels at NSD2‐enriched genes. In addition, we found that NSD2 gene was upregulated by serum stimulation and required for the induction of cell cycle‐related genes. Indeed, in both mouse and human tissues and human cancer cell lines, the expression levels of NSD2 were positively correlated with those of cell cycle‐related genes. These data reveal that NSD2 plays a pivotal role in epigenomic maintenance and cell cycle control to prevent cellular senescence. [ABSTRACT FROM AUTHOR]

The transcription factor SNAIL1 is a master regulator of epithelial‐to‐mesenchymal transition (EMT), a process entailing massive gene expression changes. To better understand SNAIL1‐induced transcriptional reprogramming we performed time‐resolved transcriptome analysis upon conditional SNAIL1 expression in colorectal cancer cells. Gene set variation analyses indicated that SNAIL1 strongly affected features related to cell cycle and Wnt/β‐Catenin signalling. This correlated with upregulation of LEF1, a nuclear binding partner of β‐Catenin. Likewise, transcriptomes of cell lines and colorectal cancers, including poor‐prognosis mesenchymal tumours, exhibit positively correlated SNAI1 and LEF1 expression, and elevated LEF1 levels parallel increased patient mortality. To delineate the functional contribution of LEF1 to SNAIL1‐induced EMT, we used the CRISPR/Cas9 system to knock‐out LEF1 in colorectal cancer cells, and to engineer cells that express LEF1 mutants unable to interact with β‐Catenin. Both complete LEF1‐deficiency and prevention of the β‐Catenin‐LEF1 interaction impaired the ability of SNAIL1 to elicit expression of an alternative set of Wnt/β‐catenin targets, and to promote cancer cell invasion. Conversely, overexpression of wildtype, but not of mutant LEF1, stimulated alternative Wnt/β‐Catenin target gene expression, and caused cell‐cycle arrest. Moreover, like SNAIL1, LEF1 retarded tumour growth in xenotransplantations. Thus, LEF1 phenocopies SNAIL1 with respect to several critical aspects of EMT. Indeed, comparative transcriptomics suggested that 35% of SNAIL1‐induced transcriptional changes are attributable to LEF1. However, LEF1 did not autonomously induce EMT. Rather, LEF1 appears to be a strictly β‐Catenin‐dependent downstream effector of SNAIL1. Apparently, SNAIL1 employs β‐Catenin‐LEF1 complexes to redirect Wnt/β‐Catenin pathway activity towards pro‐invasive and anti‐proliferative gene expression. What's new? Epithelial‐to‐mesenchymal transition (EMT), which plays a critical role in cancer progression, is underpinned by massive alterations in gene expression. While the transcription factor SNAIL1 induces many of these changes, the process of transcriptional reprogramming in EMT remains poorly understood. This study, using a colorectal cancer model, shows that SNAIL1 redirects Wnt/β‐Catenin signaling to initiate EMT. SNAIL1, via LEF1 upregulation, diverts β‐Catenin‐dependent transcription toward target genes, including genes that promote invasion and cell‐cycle arrest, despite oncogenic CTNNB1 and KRAS mutations. The findings delineate a role for β‐Catenin‐LEF1‐driven subprograms in EMT induction and show that this process includes unexpected anti‐proliferative activity. [ABSTRACT FROM AUTHOR]

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.

Summary: Background: Cell division cycle 25c (CDC25c) is a gene coding a phosphatase controlling entry into mitosis upon activation through Polo-like kinase 1 (PLK1) and serves as a key regulator of cell division. The CDC25c was reported to be dysregulated in some malignant diseases, but its role in myelofibrosis has not yet been elucidated. Methods: We have retrospectively investigated CDC25c mRNA expression in bone marrow aspirates of 43 patients with myelofibrosis (28 primary [PMF] and 15 secondary myelofibrosis [SMF]) and 12 controls. Results: CDC25c mRNA expression did not significantly differ between PMF, SMF and controls (median ∆CT 3.08 vs 2.86 vs 2.29 for PMF, SMF and controls, respectively; P = 0.162). Patients presenting with higher CDC25c mRNA expression were of older age (P = 0.037), had statistically significantly higher white-blood-cells (P = 0.017), larger liver size (P = 0.022), higher absolute neutrophil (P = 0.010), monocyte (P = 0.050), basophil (P = 0.012), and eosinophil counts (P = 0.013). Patients presenting with high CDC25c mRNA expression had statistically significantly inferior overall survival compared to low CDC25c expression group (HR = 2.99; P = 0.049). Median overall survival was not reached in patients with low and was 44 months in patients with high CDC25c expression. Conclusion: Our data suggest that higher CDC25c expression is associated with more proliferative phenotype of myelofibrosis and is prognostic of worse survival. Future studies investigating these interesting associations are warranted. [ABSTRACT FROM AUTHOR]

During CNS development, there is prominent expansion of the anterior region, the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. We find that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven ‘super-generation’ of progenitors, as well as ‘hyper-proliferation’ of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity.

Tamoxifen-induced radioresistance, reported in vitro, might pose a problem for patients who receive neoadjuvant tamoxifen treatment and subsequently receive radiotherapy after surgery. Previous studies suggested that DNA damage repair or cell cycle genes are involved, and could therefore be targeted to preclude the occurrence of cross-resistance. We aimed to characterize the observed cross-resistance by investigating gene expression of DNA damage repair genes and cell cycle genes in estrogen receptor-positive MCF-7 breast cancer cells that were cultured to tamoxifen resistance. RNA sequencing was performed, and expression of genes characteristic for several DNA damage repair pathways was investigated, as well as expression of genes involved in different phases of the cell cycle. The association of differentially expressed genes with outcome after radiotherapy was assessed in silico in a large breast cancer cohort. None of the DNA damage repair pathways showed differential gene expression in tamoxifen-resistant cells compared to wild-type cells. Two DNA damage repair genes were more than two times upregulated (NEIL1 and EME2), and three DNA damage repair genes were more than two times downregulated (PCNA , BRIP1 , and BARD1). However, these were not associated with outcome after radiotherapy in the TCGA breast cancer cohort. Genes involved in G1, G1/S, G2, and G2/M phases were lower expressed in tamoxifen-resistant cells compared to wild-type cells. Individual genes that were more than two times upregulated (MAPK13) or downregulated (E2F2 , CKS2 , GINS2 , PCNA , MCM5 , and EIF5A2) were not associated with response to radiotherapy in the patient cohort investigated. We assessed the expression of DNA damage repair genes and cell cycle genes in tamoxifen-resistant breast cancer cells. Though several genes in both pathways were differentially expressed, these could not explain the cross-resistance for irradiation in these cells, since no association to response to radiotherapy in the TCGA breast cancer cohort was found. [ABSTRACT FROM AUTHOR]

Accumulating evidence including studies of AHR-deficient mice and TCDD toxicity suggests multiple physiologic AHR functions. Challenges to identify responsible mechanisms are due to marked species differences and dependence upon cell type and cellular context. Transient AHR modulation is often necessary for physiologic functions whereas TCDD-mediated sustained receptor activation has been demonstrated to be responsible for toxic outcomes. To stimulate studies on responsible action mechanisms the commentary is focused on human AHR target genes and crosstalk with transcription factors. Discussed AHR functions include chemical and microbial defense, organ development, modulation of immunity and inflammation, reproduction, and NAD+-dependent energy metabolism. Obviously, much more work is needed to elucidate action mechanisms. In particular, studies of pathways leading to NAD+-dependent energy metabolism may shed light on the puzzling species differences of TCDD-mediated lethality and provide options for treatment of obesity and age-related degenerative diseases. [ABSTRACT FROM AUTHOR]

B-type cyclins in association with Cdk1 mediate key steps of mitosis and meiosis, by phosphorylating a plethora of substrates. Progression through the meiotic cell cycle requires the execution of two cell divisions named meiosis I and II without intervening S-phase, to obtain haploid gametes. These two divisions are highly asymmetric in the large oocyte. Chromosome segregation in meiosis I and sister chromatid segregation in meiosis II requires the sharp, switch-like inactivation of Cdk1 activity, which is brought about by degradation of B-type cyclins and counteracting phosphatases. Importantly and contrary to mitosis, inactivation of Cdk1 must not allow S-phase to take place at exit from meiosis I. Here, we describe recent studies on the regulation of translation and degradation of B-type cyclins in mouse oocytes, and how far their roles are redundant or specific, with a special focus on the recently discovered oocyte-specific role of cyclin B3. [ABSTRACT FROM AUTHOR]

Cellular checkpoints controlling entry into mitosis monitor the integrity of the DNA and delay mitosis onset until the alteration is fully repaired. However, this canonical response can weaken, leading to a spontaneous bypass of the checkpoint, a process referred to as checkpoint adaptation. Here, we have investigated the contribution of microcephalin 1 (MCPH1), mutated in primary microcephaly, to the decatenation checkpoint, a less-understood G2 pathway that delays entry into mitosis until chromosomes are properly disentangled. Our results demonstrate that, although MCPH1 function is dispensable for activation and maintenance of the decatenation checkpoint, it is required for the adaptive response that bypasses the topoisomerase II inhibition-mediated G2 arrest. MCPH1, however, does not confer adaptation to the G2 arrest triggered by the ataxia telangiectasia mutated- and ataxia telangiectasia and rad3 related-based DNA damage checkpoint. In addition to revealing a new role for MCPH1 in cell cycle control, our study provides new insights into the genetic requirements that allow cellular adaptation to G2 checkpoints, a process that remains poorly understood.--Arroyo, M., Kuriyama, R., Guerrero, I., Keifenheim, D., Cañuelo, A., Calahorra, J., Sánchez, A., Clarke, D. J., Marchal, J. A. MCPH1 is essential for cellular adaptation to the G2-phase decatenation checkpoint. [ABSTRACT FROM AUTHOR]

Solar UV radiation is a major environmental risk factor for skin cancer. Despite decades of robust and meritorious investigation, our understanding of the mechanisms underlying UV-induced skin carcinogenesis remain incomplete. We previously performed comprehensive transcriptomic profiling in human keratinocytes following exposure to different UV radiation conditions to generate UV-specific gene expression signatures. In this study, we utilized Virtual Inference of Protein Activity by Enriched Regulon (VIPER), a robust systems biology tool, on UV-specific skin cell gene signatures to identify master regulators (MRs) of UV-induced transcriptomic changes. We identified multiple prominent candidate UV MRs, including forkhead box M1 (FOXM1), thyroid hormone receptor interactor 13 and DNA isomerase II alpha, which play important roles in cell cycle regulation and genome stability. MR protein activity was either activated or suppressed by UV in normal keratinocytes. Intriguingly, many of the UV-suppressed MRs were activated in human skin squamous cell carcinomas (SCCs), highlighting their importance in skin cancer development. We further demonstrated that selective inhibition of FOXM1, whose activity was elevated in SCC cells, was detrimental to SCC cell survival. Taken together, our study uncovered novel UV MRs that can be explored as new therapeutic targets for future skin cancer treatment. [ABSTRACT FROM AUTHOR]