Prolonged Cell Cycle Research Articles

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint.

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

Evidence of 14-3-3 proteins contributing to kinetochore integrity and chromosome congression during mitosis.

The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.

Cytomegalovirus infection lengthens the cell cycle of granule cell precursors during postnatal cerebellar development.

Human cytomegalovirus (HCMV) infection in infants infected in utero can lead to a variety of neurodevelopmental disorders. However, mechanisms underlying altered neurodevelopment in infected infants remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in all regions of the brain of newborn mice, including the cerebellum. Infection resulted in disruption of cerebellar cortical development characterized by reduced cerebellar size and foliation. This disruption was associated with altered cell cycle progression of the granule cell precursors (GCPs), which are the progenitors that give rise to granule cells (GCs), the most abundant neurons in the cerebellum. In the current study, we have demonstrated that MCMV infection leads to prolonged GCP cell cycle, premature exit from the cell cycle, and reduced numbers of GCs resulting in cerebellar hypoplasia. Treatment with TNF-α neutralizing antibody partially normalized the cell cycle alterations of GCPs and altered cerebellar morphogenesis induced by MCMV infection. Collectively, our results argue that virus-induced inflammation altered the cell cycle of GCPs resulting in a reduced numbers of GCs and cerebellar cortical hypoplasia, thus providing a potential mechanism for altered neurodevelopment in fetuses infected with HCMV.

(S)-3-(3-Fluoro-4-Methoxybenzyl)-5,6,7-Trimethoxychroman-4-One Suppresses the Proliferation of Huh7 Cells by Up-regulating P21 and Inducing G2/M Phase Arrest.

Hepatocellular carcinoma (HCC) is a prevalent type of cancer worldwide. Although sorafenib is the only chemotherapy agent used for HCC, there is a need to discover a more potent anticancer agent with reduced side-effects. The compound, (S)-3-(3-fluoro-4-methoxybenzyl)-5,6,7-trimethoxychroman-4-one (FMTC), was designed to inhibit tubulin assembly but its specific mechanisms of action have not been previously investigated. Herein, we investigated the regulation mechanisms by which FMTC affects the proliferation of the HCC cell line, Huh7. The effects of FMTC on cell viability and growth were analyzed in the HCC cell line, Huh7. Cell cycle and apoptosis regulated by FMTC were analyzed using flow cytometry. To verify the regulation of mRNA and protein expression of cell proliferation-related factors by FMTC in Huh7 cells, RT-qPCR and western blot analyses were employed. FMTC suppressed cell division dose-dependently by triggering cell cycle arrest at the G2/M phase via p21 up-regulation. The increased phosphorylation of histone H3 on Ser-10 and the condensation of chromatin in FMTC-treated cells indicated mitotic arrest. Prolonged FMTC-induced cell cycle arrest triggered apoptosis. FMTC inhibits the proliferation of human liver cancer cells by up-regulating p21, thereby inducing cell cycle arrest at the G2/M phase. These findings highlight FMTC as a novel agent for HCC treatment.

TIPRL1 and its ATM-dependent phosphorylation promote radiotherapy resistance in head and neck cancer.

TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 - all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target.

Acetyl Transferase EP300 Deficiency Leads to Chronic Replication Stress in Adult T-Cell Leukemia/Lymphoma

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a disease model to study EP300 deficiency and an EP300-selective PROTAC degrader, as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, transmission of unrepaired inherited DNA lesions in the subsequent G1-phase and accumulation of cytosolic DNA in EP300-deficient cells. However, EP300 deficient cells show a significant defect in innate immune system activation. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress, persistent genomic instability and innate immune system evasion underlie aggressive chemo-resistant tumorigenesis in humans.

Narazaciclib, a Differentiated CDK4/6 Antagonist, Prolongs Cell Cycle Arrest and Metabolomic Reprogramming, Enabling Restoration of Ibrutinib Sensitivity in Btki-Resistant Mantle Cell Lymphoma

Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL. We compared the efficacy and safety profiles of narazaciclib with three health authority-approved CDKi (palbociclib, abemaciclib or ribociclib) in association with covalent (ibrutinib, acalabrutinib) and non-covalent (pirtobrutinib, ARQ-531) BTKi, across a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We integrated RNA sequencing and gene set enrichment analysis (GSEA) coupled to phospho-proteomics and kinase-substrate enrichment analysis (KSEA) to unravel the molecular bases of BTKi-CDKi drug interaction in MCL. CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, RT-PCR and western blot approaches followed by Seahorse-mediated metabolic assays were carried out for validation purposes. Finally, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in three immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft models. We found that narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC 50: 3.61 ± 2.1 µM), regardless of the sensitivity of these to ibrutinib. Although there was no correlation between CDKi sensitivity and direct repression of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response upon narazaciclib treatment. This feature was linked to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second-generation acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTKi-sensitive and BTKi-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome, although similar features were found when substituting narazaciclib with abemaciclib. The integration of transcriptomic and phospho-proteomic synergistic signatures further revealed that narazaciclib-ibrutinib combo modulated signatures related to DNA repair, P53 signaling, and glycolytic activity. Accordingly, in this model, narazaciclib/ibrutinib-exposed cells underwent a metabolic switch from glycolytic to oxidative phosphorylation (OxPhos) phenotype accompanied by intracellular accumulation of mitochondria and increased mtDNA copy number. This switch appeared to be specific to MCL cells rendered refractory to ibrutinib upon activation of alternative NFkB signaling, as it was not found in MCL-BTKmut cells. In vivo, while narazaciclib single agent achieved a moderate TGI in the CAM model, the narazaciclib/ibrutinib combo reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into host bone marrow, with no detectable toxicity. In conclusion, our findings demonstrate that narazaciclib is safe and effective as a single agent in preclinical models of MCL, including BTKi-resistant cases. Its combination with ibrutinib achieved a synergistic tumoricidal effect in vitro and in vivo, accelerating cell cycle blockade and reverting the metabolic reprogramming characterizing MCL refractoriness to BTKi therapy.

Ras-Related Nuclear Protein-Binding Protein 9 Regulates Gastric Cancer Cell Cycle and Apoptosis Through the PTEN/PI3K/AKT Signaling Pathway

We investigated the impact of RanBP9 on cell cycle progression and apoptosis in gastric cancer cells. RanBP9 expression was analyzed in 38 clinical gastric cancer tissues using Western blotting. Lentiviral transfection was utilized to establish GES-1 gastric cancer cell models that either overexpressed or silenced RanBP9. Cell proliferation and apoptosis were assessed using MTT and TUNEL staining assays, respectively. Apoptosis-related factors were analyzed by Western blotting and qRT-PCR. Flow cytometry and qRT-PCR were employed to evaluate cell cycle progression and the mRNA levels of CDK4/CyclinD1. The PTEN/PI3K/AKT signaling pathway was examined by Western blotting. We observed a significant reduction in RanBP9 expression in gastric cancer tissues. Overexpression of RanBP9 in GES-1 cells suppressed cell activity, enhanced apoptosis, increased Caspase3 expression and the Bax/Bcl-2 ratio, and decreased CDK4 and CyclinD1 expression, thereby preventing S phase entry. Conversely, knockdown of RanBP9 yielded opposite results. Furthermore, we found that RanBP9 negatively regulated the PTEN/PI3K/AKT pathway. Our findings demonstrate low expression of RanBP9 in gastric cancer tissues and cell lines. We have also established that RanBP9 negatively regulates the PTEN/PI3K/AKT pathway, resulting in cell cycle prolongation and promotion of apoptosis in GES-1 cells.

Functional inhibition of RECQL5 helicase elicits non-homologous end joining response and sensitivity of breast cancers to PARP inhibitor.

Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous recombination (HR) deficiency, based on synthetic lethality concept. However, ∼90% of breast cancers are BRCA-wild type; they repair PARPi mediated damage through HR, leading to intrinsic de novo resistance. Hence, there is an unmet need of exploring novel targets in HR-proficient aggressive breast cancers for PARPi treatment. RECQL5 physically interacts and disrupts RAD51 from pre-synaptic filaments, aiding HR resolution, replication fork protection and preventing illegitimate recombination. In the current investigation, we show that targeted inhibition of HR by stabilization of RAD51-RECQL5 complex by a pharmacological inhibitor of RECQL5 (4a; 1,3,4-oxadiazole derivative) in the presence of PARPi [talazoparib (BMN673)] leads to abolition of functional HR with uncontrolled activation of NHEJ repair. This was assessed by GFP based NHEJ reporter assay, KU80 recruitment and in vitro NHEJ based plasmid ligation assay. Concomitant treatment with talazoparib and 4a generates copious amounts of replication stress, prolonged cell cycle arrest, extensive double strand breaks (DSBs) and mitotic catastrophe, leading to sensitization of HR-proficient breast cancers. Suppression of NHEJ activity abolishes 4a-mediated sensitization of breast cancers to PARPi treatment. Imperatively, 4a was ineffective against normal mammary epithelial cells, which expresses low RECQL5 vis-à-vis breast cancer cells. Moreover, functional inhibition of RECQL5 suppresses metastatic potential of breast cancer cells in response to PARPi. Together, we identified RECQL5 as a novel pharmacological target for expanding PARPi based treatment horizon for HR-proficient cancers.

Ribociclib and endocrine therapy as adjuvant treatment in patients with HR+/HER2- early breast cancer: Primary results from the phase III NATALEE trial.

LBA500 Background: RIB + ET has demonstrated significant survival benefits in pre- and postmenopausal pts with HR+/HER2− metastatic BC. To investigate whether RIB + ET also improves outcomes in early BC (EBC), the Phase III NATALEE trial (NCT03701334) evaluated adjuvant RIB + ET in a broad population of pts with stage II or III HR+/HER2− EBC at risk for recurrence, including pts with no nodal involvement (N0). As extended duration of tx is crucial to prolong cell cycle arrest and drive more tumor cells into senescence or death, a 3-y duration of RIB tx at a dose of 400 mg was chosen to improve tolerability while maintaining efficacy. Results from a prespecified interim analysis of invasive disease–free survival (iDFS; primary endpoint) are presented. Methods: Men and pre- or postmenopausal women were randomized 1:1 to RIB (400 mg/day; 3 wk on/1 wk off for 3 y) + ET (letrozole 2.5 mg/day or anastrozole 1 mg/day, for ≥ 5 y) or ET alone. Men and premenopausal women also received goserelin. Eligible pts had an ECOG PS of 0-1 and BC anatomic stage IIA (either N0 with additional risk factors or 1-3 axillary lymph nodes [N1]), stage IIB, or stage III per AJCC (8th ed); prior (neo)adjuvant ET was allowed if initiated ≤ 12 mo before randomization. Stratification factors were menopausal status, disease stage, prior (neo)adjuvant chemotherapy, and geographic region. This prespecified interim analysis of iDFS, defined per STEEP criteria, was planned after ≈ 425 iDFS events (≈ 85% of planned total events). iDFS was evaluated by Kaplan-Meier methods, and statistical comparison was made by a stratified log-rank test, with a protocol-defined Lan-DeMets (O'Brien-Flemming) stopping boundary of a 1-sided P < .0128 for superior efficacy. Results: From 10 Jan 2019 to 20 April 2021, 5101 pts were randomized (RIB+ET, n = 2549; ET alone, n = 2552). As of the data cutoff (11 Jan 2023), median follow-up was 34 mo (min, 21 mo). 3- and 2-y RIB tx was completed by 515 pts (20.2%) and 1449 pts (56.8%), respectively; 3810 (74.7%) remained on study tx (RIB+ET, n = 1984; ET alone, n = 1826). iDFS was evaluated after 426 events (RIB + ET, n = 189; ET alone, n = 237). RIB + ET demonstrated significantly longer iDFS than ET alone (HR, 0.748; 95% CI, 0.618-0.906; P = .0014); 3-y iDFS rates were 90.4% vs 87.1%. iDFS benefit was generally consistent across stratification factors and other subgroups. Secondary endpoints of overall survival, recurrence-free survival, and distant disease–free survival consistently favored RIB. RIB at 400 mg had a favorable safety profile with no new signals. Conclusions: Ribociclib added to standard-of-care ET demonstrated a statistically significant, clinically meaningful improvement in iDFS with a well-tolerated safety profile. The NATALEE results support ribociclib + ET as the treatment of choice in a broad population of pts with stage II or III HR+/HER2− EBC, including pts with N0 disease. Clinical trial information: NCT03701334 .

Prolonged cell cycle arrest by the CDK4/6 antagonist narazaciclib restores ibrutinib response in preclinical models of BTKi‐resistant mantle cell lymphoma

Introduction: Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL models. Methods: We compared the efficacy and safety of narazaciclib with the health authority approved CDKi in association with different BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We used the CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, and RNA sequencing followed by gene set enrichment analysis (GSEA), RT-PCR and western blot validations. In addition, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in an immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft. Results: Narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC50: 3.61 ± 2.1 µM), regardless of their sensitivity to ibrutinib. Although there was no correlation between CDKi sensitivity and activation of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response (NES > 2.5) upon narazaciclib treatment. This feature was associated to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second generation therapeutic acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTK-sensitive and BTK-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant (+10%–15%) augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome. Both the downregulation of phospho-histone H3 and the upregulation of p-p27/p27 and p16, also underwent a 10%–15% improvement in combination-treated cells. In vivo, while narazaciclib single agent achieved a 28% TGI in the CAM model, the narazaciclib-ibrutinib combination reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into the bone marrow, with no detectable toxicity. Conclusions: Narazaciclib, due to its completely distinct MoA from BTKi involving the direct modulation of the cell cycle, can achieve significant synergistic activity with ibrutinib in vitro and in vivo, especially in BTKi-resistant models of MCL. Ongoing phospho-proteomics and genetic edition assays will help deciphering the molecular bases of this unique drug cooperation at the cell cycle level. Encore Abstract - previously submitted to AACR 2023 The research was funded by: This study was financially supported by Onconova Therapeutics, Spanish Ministry of Science and Innovation (grant PID2021-123039OB-C21) (to GR). MFS holds a predoctoral fellowship (FI19/00338) from Instituto de Salud Carlos III. This work was carried out under the CERCA Program (Generalitat de Catalunya). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. A. Makovski-Silverstein Employment or leadership position: Onconova Therapeutics S. Cosenza Employment or leadership position: Onconova Therapeutics G. Roué Research funding: Onconova Therapeutics

Alternative CDC20 translational isoforms tune mitotic arrest duration.

Mitotic defects activate the spindle-assembly checkpoint, which inhibits the anaphase-promoting complex co-activator CDC20 to induce a prolonged cell cycle arrest1,2. Once errors are corrected, the spindle-assembly checkpoint is silenced, allowing anaphase onset to occur. However, in the presence of persistent unresolvable errors, cells can undergo 'mitotic slippage', exiting mitosis into a tetraploid G1 state and escaping the cell death that results from a prolonged arrest. The molecular logic that enables cells to balance these duelling mitotic arrest and slippage behaviours remains unclear. Here we demonstrate that human cells modulate the duration of their mitotic arrest through the presence of conserved, alternative CDC20 translational isoforms. Downstream translation initiation results in a truncated CDC20 isoform that is resistant to spindle-assembly-checkpoint-mediated inhibition and promotes mitotic exit even in the presence of mitotic perturbations. Our study supports a model in which the relative levels of CDC20 translational isoforms control the duration of mitotic arrest. During a prolonged mitotic arrest, new protein synthesis and differential CDC20 isoform turnover create a timer, with mitotic exit occurring once the truncated Met43 isoform achieves sufficient levels. Targeted molecular changes or naturally occurring cancer mutations that alter CDC20 isoform ratios or its translational control modulate mitotic arrest duration and anti-mitotic drug sensitivity, with potential implications for the diagnosis and treatment of human cancers.

The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved.

Human papillomavirus associated XPF deficiency increases alternative end joining and cisplatin sensitivity in head and neck squamous cell carcinoma

ObjectivesHuman papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) showed a considerably better prognosis with greater cisplatin sensitivity compared to their HPV-negative counterparts. Deciphering the underlying molecular mechanisms for HPV-induced cisplatin sensitivity is imperative to improve the prognosis of HPV-negative HNSCC. Materials and MethodsThe Fanconi anemia (FA) pathway status in HNSCC cells was analysed by detecting the cell cycle and chromosomal aberrations. XPF expression was validated using PCR, western blot, and immunohistochemistry. Droplet digital PCR and GFP expressing reporter assay were used to analyse the changes in alternative end-joining (alt-EJ) levels. The cisplatin sensitization was verified by cell proliferation assay, clonogenic cell survival assay, and TUNEL. ResultsHPV-positive HNSCC cells showed significant prolonged G2-M cell cycle arrest and aberrant chromosome formation under interstrand crosslinker treatment. Both mRNA and protein expression of XPF were considerably decreased in HPV-positive HNSCC, according to the analysis of cellular and clinical data. XPF inhibition upregulated the activity of the alt-EJ pathway in HPV-negative HNSCC cells by 32.02% (P < 0.001) but had little effect on HPV-positive HNSCC. Consistent with this, simultaneous suppression of XPF and alt-EJ enhanced cisplatin sensitivity of HPV-negative HNSCC in vitro and in vivo. ConclusionHPV-positive HNSCC cells exhibit a profound FA pathway deficiency associated with reduced XPF expression. HNSCC cells with compromised XPF function are more reliant on the alt-EJ pathway for genomic stability. Combining FA and alt-EJ inhibition may be used to cope with the hard-to-treat HPV-negative HNSCC.

Changes in the Proteome Profile of A549 Cells Following Helichrysetin-Induced Apoptosis Suggest the Involvement of DNA Damage Response and Cell Cycle Arrest-Associated Proteins.

Our previous findings demonstrated that Helichrysetin possessed promising anti-cancer activity. It was able to induce apoptosis in the A549 cell line. However, its mechanism of action is unknown. The present study aimed to unravel possible underlying molecular mechanisms of helichrysetin-induced apoptosis in A549 (human lung carcinoma) cells using comparative quantitative proteomics (iTRAQ labeled), followed by an exhaustive bioinformatics analysis. Our results suggested that DNA damage response (DDR) and cell cycle arrest were responsible for lung cancer cell death with helichrysetin treatment. Among proteins that changed in abundance were Nrf2 and HMOX1. They are oxidative stress-related proteins and were increased in abundance. BRAT1 was also increased in abundance, suggesting an increase in DNA damage repair, indicating the occurrence of DNA damage due to oxidative stress. However, several essential DDR downstream proteins such as p-ATM, BRCA1, FANCD2, and Rb1 that would further increase DNA damage were found to be dramatically decreased in relative abundance. Cell cycle-related proteins, p53, p21, and cyclin D1, were increased while cyclin A, cyclin E, and cdk2 were decreased. This is predicted to facilitate S-phase arrest. Furthermore, excessive DNA damage and prolonged arrest would in turn result in the induction of mitochondrial-mediated apoptosis. Based on these observations, we postulate that the effects of helichrysetin were in part via the suppression of DNA damage response which led to DNA damage and prolonged cell cycle arrest. Subsequently, this event initiated mitochondrial-mediated apoptosis in A549 lung cancer cells.

Detection of De Novo Dividing Stem Cells In Situ through Double Nucleotide Analogue Labeling.

Tissue-specific somatic stem cells are characterized by their ability to reside in a state of prolonged reversible cell cycle arrest, referred to as quiescence. Maintenance of a balance between cell quiescence and division is critical for tissue homeostasis at the cellular level and is dynamically regulated by numerous extrinsic and intrinsic factors. Analysis of the activation of quiescent stem cells has been challenging because of a lack of methods for direct detection of de novo dividing cells. Here, we present and experimentally verify a novel method based on double labeling with thymidine analogues to detect de novo dividing stem cells in situ. In a proof of concept for the method, we show that memantine, a drug widely used for Alzheimer's disease therapy and a known strong inducer of adult hippocampal neurogenesis, increases the recruitment into the division cycle of quiescent radial glia-like stem cells-primary precursors of the adult-born neurons in the hippocampus. Our method could be applied to assess the effects of aging, pathology, or drug treatments on the quiescent stem cells in stem cell compartments in developing and adult tissues.

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2´-deoxyuridine (EdU) incorporation assay.

Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial-mesenchymal transition in clear-cell renal cell carcinoma.

RUNX3 is a transcription factor and tumor suppressor that is silenced or inactivated in diverse tumors. The effect of RUNX3 on the epithelial-mesenchymal transition in clear-cell renal cell carcinoma (CCRCC) remains unclear. We determined the expression of RUNX3 and E-cadherin in tumor tissues and adjacent normal tissues of 30 CCRCC patients; established cultured CCRCC cells with the overexpression of RUNX3; and examined the in vivo tumorigenic function of RUNX3 in a nude mouse xenograft model of CCRCC. RUNX3 and E-cadherin were downregulated in human CCRCC samples. Cell lines with RUNX3 overexpression had reduced cell proliferation, invasion, and migration, a prolonged cell cycle, increased apoptosis, and increased expression of E-cadherin. In the nude mouse xenograft model of CCRCC, tumors with the overexpression of RUNX3 had smaller volumes and weights and had increased expression of E-cadherin. In conclusion, RUNX3 overexpression increased the level of E-cadherin and inhibited the proliferation, invasion, and migration of CCRCC in vitro and in vivo. RUNX3 has potential use as a biomarker for prognostic monitoring of CCRCC and as a therapeutic target for the treatment of this cancer.

DOT1L regulates chamber-specific transcriptional networks during cardiogenesis and mediates postnatal cell cycle withdrawal

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

Revealing microcystin-LR ecotoxicity to earthworm (Eisenia fetida) at the intestinal cell level

Potential adverse effects of microcystin-LR (MC-LR) on soil invertebrates have not been studied. Here we investigated the mechanism of MC-LR toxicity to earthworm (Eisenia fetida) intestine at the individual level and at the cellular level. The results showed an inverse relationship between the bodyweight and survival rate of earthworms over exposure time- and MC-LR doses in soil. Dose-dependent intestinal lesions and disturbances of enzymatic activities (e.g., cellulase, Na+/K+-ATPase, and AChE) were observed, which resulted in intestinal dysfunction. Excessive reactive oxygen species generation led to DNA damage and lipid peroxidation of intestinal cells. The oxidative damage to DNA prolonged cell cycle arrest at the G2/M-phase transition in mitosis, thus stimulating and accelerating apoptosis in earthworm intestine. MC-LR target earthworm intestine tissue. MC-LR at low concentrations can damage earthworm intestine regardless of exposure routes (oral or contact). High toxicity of MC-LR to earthworms delineates its ecological risks to terrestrial ecosystems.