G2 Delay Research Articles

Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network

Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing invivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.

Using Live Imaging and Fluorescence Ubiquitinated Cell Cycle Indicator Embryonic Stem Cells to Distinguish G1 Cell Cycle Delays for General Stressors like Perfluoro-Octanoic Acid and Hyperosmotic Sorbitol or G2 Cell Cycle Delay for Mutagenic Stressors like Benzo(a)pyrene.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 μM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

Using Live Imaging and FUCCI Embryonic Stem Cells to Rank DevTox Risks: Adverse Growth Effects of PFOA Compared With DEP Are 26 Times Faster, 1,000 Times More Sensitive, and 13 Times Greater in Magnitude.

Fluorescent ubiquitination-based cell cycle indicator (FUCCI) embryonic stem cells (ESCs), which fluoresce green during the S-G2-M phases, generate an S-shaped curve for the accumulation of cells during normal stemness (NS) culture with leukemia-inhibitory factor (LIF). Since it was hypothesized that a culture of ESCs was heterogeneous in the cell cycle, it was expected that increased S-G2-M-phases of the cell cycle would make an S-shaped curve parallel to the accumulation curve. Unexpectedly, it was observed that the fraction of FUCCI ESCs in green decreases over time to a nadir at ∼24 h after previous feeding and then rapidly enters S-G2-M-phases after medium change. G1 delay by infrequent medium change is a mild stress, as it does not affect growth significantly when frequency is increased to 12 h. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) were used as examples of members of the per- and polyfluoroalkyl substances (PFAS) and phthalate families of chemicals, respectively. Two adverse outcomes were used to compare dose- and time-dependent effects of PFOA and DEP. The first was cell accumulation assay by time-lapse confluence measurements, largely at Tfinal/T74 h. The second was by quantifying dominant toxicant stress shown by the suppression of mild stress that creates a green fed/unfed peak. In terms of speed, PFOA is 26 times faster than DEP for producing a time-dependent LOAEL dose at 100 uM (that is, 2 h for PFOA and 52 h for DEP). PFOA has 1000-fold more sensitive LOAEL doses than DEP for suppressing ESC accumulation (confluence) at day 3 and day 2. There were two means to compare the magnitude of the growth suppression of PFOA and DEP. For the suppression of the accumulation of cells measured by confluence at Tfinal/T74h, there was a 13-fold suppression at the highest dose of PFOA > the highest dose of DEP. For the suppression of entry into the cell cycle after the G1 phase by stress on day 1 and 2, there is 10-fold more suppression by PFOA than DEP. The data presented here suggest that FUCCI ESCs can assay the suppression of accumulated growth or predict the suppression of future growth by the suppression of fed/unfed green fluorescence peaks and that PFOA’s adverse effects are faster and larger and can occur at more sensitive lower doses than DEP.

Resistance to the CHK1 inhibitor prexasertib involves functionally distinct CHK1 activities in BRCA wild-type ovarian cancer

High grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy in BRCA wild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1’s regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.

Hydrogen Peroxide Mediates Artemisinin-Derived C-16 Carba-Dimer-Induced Toxicity of Human Cancer Cells.

This study used a nitroaliphatic chemistry approach to synthesize a novel artemisinin-derived carba-dimer (AG-1) and determined its anti-proliferative effects in human normal and cancer cells. AG-1 treatments selectively inhibit proliferation of cancer cells compared to normal human fibroblasts. Compared to artemisinin, AG-1 is more toxic to human breast, prostate, head–neck, pancreas and skin cancer cells; 50% inhibition (IC50) 123 µM in AG-1 vs. 290 µM in artemisinin-treated breast cancer cells. AG-1 treatment decreased (~5 folds) cyclin D1 protein expression that correlated with an increase in the percentage of cells in the G1-phase, suggesting a G1 delay. AG-1-induced toxicity was independent of the DNA damage at 72 h post-treatment, as measured by micronuclei frequency and γH2AX protein levels. Results from electron paramagnetic resonance spectroscopy showed Fe-catalyzed formation of AG-1 carbon-centered radicals in a cell-free system. Flow cytometry analysis of H2DCF-DA oxidation showed a significant increase in the steady-state levels of reactive oxygen species (ROS) in AG-1-treated cells. Pre-treatment with N-acetyl-l-cysteine and antioxidant enzymes (superoxide dismutase and catalase) significantly suppressed AG-1-induced toxicity, suggesting that superoxide and hydrogen peroxide contribute to AG-1-induced toxicity in human cancer cells. AG-1 represents a novel class of anti-cancer drug that is more potent than its parent compound, artemisinin.

Effects of Grape Seed Extract and Proanthocyanidin B2 on In Vitro Proliferation, Viability, Steroidogenesis, Oxidative Stress, and Cell Signaling in Human Granulosa Cells.

Reactive oxygen species (ROS) which lead to oxidative stress affect ovarian function. Grape seed extract (GSE) could be proposed as an effective antioxidant, particularly due to its proanthocyanidin content. In this study, we investigated a dose effect (0, 0.01, 0.1, 1, 10, 50, and 100 μg/mL) of GSE and proanthocyanidin B2 (GSPB2) on the ROS content, cell proliferation, cell viability, and steroidogenesis in both primary luteinized granulosa cells (hGC) and the tumor granulosa cell line (KGN). The levels of ROS were measured using ROS-Glo assay. Cell proliferation and viability were evaluated by [3H]-thymidine incorporation and Cell Counting Kit-8 (CCK8) assay, respectively. Steroid secretion was evaluated by radioimmunoassay. We also analyzed the cell cycle component protein level and signaling pathways by immunoblot and the NOX4 mRNA expression by RTqPCR. From 0.1 to 1 μg/mL, GSE and GSBP2 reduced the ROS cell content and the NOX4 mRNA levels, whereas, GSE and GSBP2 increased the ROS cell content from 50 to 100 μM in both hGC and KGN. GSE and GSPB2 treatments at 50 and 100 μg/mL induced a delay in G1 to S phase cell cycle progression as determined by fluorescence-activated cell sorting. Consequently, they reduced cell growth, cyclin D2 amount, and Akt phosphorylation, and they increased protein levels of p21 and p27 cyclin-dependent kinase inhibitors. These data were also associated with an increase in cell death that could be due to a reduction in Bcl-2-associated death promoter (BAD) phosphorylation and an increase in the cleaved-caspase-3 level. All these negative effects were not observed at lower concentrations of GSE and GSPB2 (0.01 to 10 μg/mL). Interestingly, we found that GSE and GSPB2 treatments (0.1 to 100 μg/mL) improved progesterone and estradiol secretion and this was associated with a higher level of the cholesterol carriers, StAR (steroidogenic acute regulatory protein), CREB (Cyclic adenosine monophosphate Response Element-binding protein), and MAPK ERK1/2 (Mitogen-Activated Protein Kinases Extracellular signal-Regulated Kinases 1/2) phosphorylation in both hGC and KGN cells. Taken together, GSE and GSPB2 (0.1–10 μg/mL) in vitro treatments decrease oxidative stress and increase steroidogenesis without affecting cell proliferation and viability in human granulosa cells.

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast.

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.

Caffeine Sensitizes U87-MG Human Glioblastoma Cells to Temozolomide through Mitotic Catastrophe by Impeding G2 Arrest.

Temozolomide (TMZ) is the first-line chemotherapeutic agent in the treatment of glioblastoma multiforme (GBM). Despite its cytotoxic effect, TMZ also induces cell cycle arrest that may lead to the development of chemoresistance and eventual tumor recurrence. Caffeine, a widely consumed neurostimulant, shows anticancer activities and is reported to work synergistically with cisplatin and camptothecin. The present study aimed to investigate the effects and the mechanisms of action of caffeine used in combination with TMZ in U87-MG GBM cells. As anticipated, TMZ caused DNA damage mediated by the ATM/p53/p21 signaling pathway and induced significant G2 delay. Concurrent treatment with caffeine repressed proliferation and lowered clonogenic capacity on MTT and colony formation assays, respectively. Mechanistic study showed that coadministration of caffeine and TMZ suppressed the phosphorylation of ATM and p53 and downregulated p21 expression, thus releasing DNA-damaged cells from G2 arrest into premature mitosis. Cell cycle analysis demonstrated that the proportion of cells arrested in G2 phase decreased when caffeine was administered together with TMZ; at the same time, the amount of cells with micronucleation and multipolar spindle poles increased, indicative of enhanced mitotic cell death. Pretreatment of cells with caffeine further enhanced mitotic catastrophe development in combined treatment and sensitized cells to apoptosis when followed by TMZ alone. In conclusion, our study demonstrated that caffeine enhanced the efficacy of TMZ through mitotic cell death by impeding ATM/p53/p21-mediated G2 arrest.

Yeast Cip1 is activated by environmental stress to inhibit Cdk1\u2013G1 cyclins via Mcm1 and Msn2/4

Upon environmental changes, proliferating cells delay cell cycle to prevent further damage accumulation. Yeast Cip1 is a Cdk1 and Cln2-associated protein. However, the function and regulation of Cip1 are still poorly understood. Here we report that Cip1 expression is co-regulated by the cell-cycle-mediated factor Mcm1 and the stress-mediated factors Msn2/4. Overexpression of Cip1 arrests cell cycle through inhibition of Cdk1–G1 cyclin complexes at G1 stage and the stress-activated protein kinase-dependent Cip1 T65, T69, and T73 phosphorylation may strengthen the Cip1and Cdk1–G1 cyclin interaction. Cip1 accumulation mainly targets Cdk1–Cln3 complex to prevent Whi5 phosphorylation and inhibit early G1 progression. Under osmotic stress, Cip1 expression triggers transient G1 delay which plays a functionally redundant role with another hyperosmolar activated CKI, Sic1. These findings indicate that Cip1 functions similarly to mammalian p21 as a stress-induced CDK inhibitor to decelerate cell cycle through G1 cyclins to cope with environmental stresses.

ESE-1/ELF3 mRNA expression associates with poor survival outcomes in HER2+ breast cancer patients and is critical for tumorigenesis in HER2+ breast cancer cells.

ESE-1/Elf3 and HER2 appear to establish a positive feedback regulatory loop, but the precise role of ESE-1 in HER2+ breast tumorigenesis remains unknown. Analyzing public repositories, we found that luminal B and HER2 subtype patients with high ESE-1 mRNA levels displayed worse relapse free survival. We stably knocked down ESE-1 in HER2+ luminal B BT474 cells and HER2 subtype SKBR3 cells, which resulted in decreased cell proliferation, colony formation, and anchorage-independent growth in vitro. Stable ESE-1 knockdown inhibited HER2-dependent signaling in BT474 cells and inhibited mTOR activation in SKBR3 cells, but reduced Akt signaling in both cell types. Expression of a constitutively-active Myr-Akt partially rescued the anti-proliferative effect of ESE-1 knockdown in both cell lines. Furthermore, ESE-1 knockdown inhibited cyclin D1, resulting in a G1 delay in both cell lines. Finally, ESE-1 knockdown completely inhibited BT474 cell xenograft tumors in NOD/SCID female mice, which correlated with reduced in vitro tumorsphere formation. Taken together, these results reveal the ESE-1 controls transformation via distinct upstream signaling mechanisms in SKBR3 and BT474 cells, which ultimately impinge on Akt and cyclin D1 in both cell types to regulate cell proliferation. Particularly significant is that ESE-1 controls tumorigenesis and is associated with worse clinical outcomes in HER2 breast cancer.

The Translationally Controlled Tumor Protein and the Cellular Response to Ionizing Radiation-Induced DNA Damage.

The absorption of ionizing radiation by living cells can directly disrupt atomic structures, producing chemical and biological changes. It can also act indirectly through radiolysis of water, thereby generating reactive chemical species that may damage nucleic acids, proteins, and lipids. Together, the direct and indirect effects of radiation initiate a series of biochemical and molecular signaling events that may repair the damage or culminate in permanent physiological changes or cell death. In efforts to gain insight into the mechanisms underlying these effects, we observed a prominent upregulation of the Translationally Controlled Tumor Protein (TCTP) in low dose/low dose rate 137Cs γ-irradiated cells that was associated with adaptive responses that reduced chromosomal damage to a level lower than what occurs spontaneously. Therefore, TCTP may support the survival and genomic integrity of irradiated cells through a role in the DNA damage response. Consistent with this postulate, TCTP was shown to physically interact with ATM, an early sensor of DNA damage, and to exist in a complex with γH2A.X, in agreement with its distinct localization with the foci of the DNA damage marker proteins γH2A.X, 53BP1, and P-ATM. Cells lacking TCTP failed to repair chromosomal damage induced by γ-rays. Further, TCTP was shown to interact with the DNA-binding subunits, Ku70 and Ku80, of DNA-PK, a major participant in nonhomologous end joining of DNA double strand breaks. Moreover, TCTP physically interacted with p53, and its knockdown attenuated the radiation-induced G1 delay, but prolonged the G2 delay. Here, we briefly review the biochemical events leading to DNA damage by ionizing radiation and to its sensing and repair, and highlight TCTP's critical role in maintaining genomic integrity in response to DNA-damaging agents.

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma

ABSTRACTDiffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

Abstract 1098: MiR-203 suppresses cutaneous squamous cell carcinoma growth and targets the myc oncogene

Abstract Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer in man and accounts for approximately 20% of non-melanoma skin cancers. Although most cSCC are benign, poorly differentiated cSCC poses a significant risk of metastasis and death. To date, little is known about the difference in molecular background between low-risk and high risk cSCC. MicroRNAs are short regulatory RNAs that can regulate gene expression and cellular functions. Here we demonstrate for the first time that the expression of miR-203 in cSCC correlates with tumor differentiation grade, being down-regulated in poorly but not in moderately or well differentiated cSCC. In vitro, miR-203 causes a delay in G1 to S phase transition and suppresses cell proliferation in human cSCC cells. Furthermore, miR-203 suppresses scratch-wound closure, cell migration, cell invasion, colony forming ability and angiogenesis-inducing capacity of cSCC cells. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 reveals dramatic changes in gene networks related to carcinogenesis, with significant suppression of genes with known oncogenic functions (e.g. PCNA, EGFR, HGF). Using luciferase reporter assays and site-specific mutagenesis, we identify c-MYC as a novel target of miR-203. Highlighting the importance of c-MYC within miR-203-regulated gene network, in rescue experiments overexpression of c-MYC reverses miR-203-induced growth arrest in cSCC. In vivo, overexpression of miR-203 in cSCC cell lines result in reduced xenograft tumor volume and decreased vessel density. Together our data show that miR-203 acts a tumor suppressor in cSCC, affecting several oncogenic and angiogenic mechanisms. Importantly, its restoration may provide therapeutic benefit in particular in poorly differentiated cSCC. Citation Format: Warangkana Lohcharoenkal, Masako Harada, Jakob Lovén, Florian Meisgen, Ning Xu Landén, Lingyun Zhang, Liisa Nissinen, Veli-Matti Kähäri, Mona Ståhle, Enikö Sonkoly, Dan Grander, Marie Arsenian-Henriksson, Andor Pivarcsi. MiR-203 suppresses cutaneous squamous cell carcinoma growth and targets the myc oncogene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1098.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression.

The anaphase-promoting complex works together with the SCF complex for proteolysis of the S-phase cyclin Clb6 during the transition from G1 to S phase

In Saccharomyces cerevisiae, the S-phase cyclin Clb6 is expressed shortly before the G1/S transition. It has been shown that in S phase the SCFCdc4 ubiquitin ligase controls Clb6 proteolysis, which requires cyclin-dependent kinases activity. A Clb6-3A mutant, bearing non-phosphorylatable mutations at S6A, T39A, and S147A, was observed to be hyperstabilized in S-phase but was unstable in mitosis. In this study, we found that the APCCdh1 form of the Anaphase-Promoting Complex (APC) was required for Clb6 proteolysis in both early and late G1. An in vitro ubiquitination assay confirmed that Clb6 is a substrate for APCCdh1. A KEN box and a destruction box in the Clb6N-terminus were identified. Mutations in the KEN box (mkb) and/or the destruction box (mdb) enhanced Clb6 stability in G1. Expression of Clb6mkd, bearing both mutations in the mkb and mdb, allowed cells to bypass the late G1 arrest caused by cdc4-1. This bypass phenotype was observed to depend upon CDK phosphorylation at residues S6, T39 and S147. Compared to Clb6, overexpression of Clb6ST, bearing all five mutations of S6A, T39A, S147A, mkb and mdb in combination, had a greater effect on promoting expression of Clb2 and S-phase entry, caused a greater G2 delay and a greater defect in cell division. Swe1 was also required for bud emergence when Clb6ST was overexpressed. Our observations suggest that both APCCdh1 and SCFCdc4-dependent proteolysis of Clb6 at the G1/S border are crucial for multiple cell cycle regulated events including proper expression of Clb2, the G1/S and G2/M cell cycle transitions and for proper completion of cell division at mitotic exit.

RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

The present study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. We found that deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. We found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins.

Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.

Abstract 3777: In silico rendering of cell cycle progression of erlotinib and gemcitabine treatment in pancreatic cancer cells

Abstract The association of erlotinib (ERL) with gemcitabine (GEM) is a promising combined therapy for advanced pancreatic cancer, but current protocols have only a modest impact on the overall survival of patients respect to GEM alone. Sequential treatment can improve the efficacy of this drug combination. Towards optimization of the drug combination, we simulated cell cycle progression during/after treatment, based on the dynamics of checkpoint activation, with the probabilities of cell arrest or death in each cell cycle phase, applying a previously established experimental/computational approach (Lupi et al. Cancer Res. 64: 2825; Ubezio et al Cancer Res. 69: 5234; Falcetta et al. PLoS Comput Biol. e1003293). The simulation gives the temporal evolution of cell flow through G1, S and G2M over subsequent division cycles, from which the time courses of cell cycle percentages and the number of cells in each cell generation were derived enabling a joint validation of the model with independent flow cytometric and time-lapse live-imaging experimental data, collected in time course experiments up to 96h with different concentrations of GEM (6h treatment) and ERL (48h treatment) with two pancreatic cell lines (BxPC-3 and capan-1). Analysis of the response to ERL treatment reveals that at cytostatic concentrations a partial G1 arrest mainly occurs after the first division following the start of treatment (gen 1 cells) while a stronger delay of the progression in S phase is active also before division (gen 0). The activity of G1 and S phase checkpoints were dose dependent and at higher cytotoxic concentrations we observed a near complete arrest in both G1 and S phases while most cell deaths were in phase S in gen 0. After treatment discontinuation, while G1 blocked cells re-enter in cycle, S phase progression in gen 0 and gen 1 remained slow but without additional death events. The model of GEM demonstrated the activation of a strong delay in phase S shortly after the start of treatment, both in gen 0 and gen 1, already at non-cytotoxic concentrations, lasting several hours after treatment discontinuation, and thereafter progressively reduced, resulting in a wave of broadly semisynchronised cells traversing S and G2M phases in gen 0. At cytotoxic concentrations, part of the cells remained arrested in S phase and eventually died. A temporary delay in G1 was also demonstrated already at fairly low concentrations, acting only during exposure to the drug. Based on these models of GEM and ERL treatment, studies are ongoing to simulate the expected outcomes of sequential treatments. Preliminary analysis suggests that the GEM = > ERL sequence is particularly favourable in both cell lines when a cytostatic ERL concentration was given after a fairly cytototoxic GEM concentration when S phase progression resumed, by strengthening the long term cytostatic effects in S phase. Citation Format: Monica Lupi, Francesca Falcetta, Maurizio D'Incalci, Paolo Ubezio. In silico rendering of cell cycle progression of erlotinib and gemcitabine treatment in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3777. doi:10.1158/1538-7445.AM2015-3777

Clinicopathological significance of ARID1B in breast invasive ductal carcinoma.

Identification of prognostic and predictive biomarkers for breast cancer is essential to better stratify patients for treatment and evaluate patient outcome. AT-rich interactive domain-containing protein 1B (ARID1B) is implicated in cell proliferation, but its role in tumorigenesis remains unclear. Immunohistochemical analysis of ARID1B expression using breast cancer tissue microarrays containing 156 breast invasive ductal carcinoma patient samples and subsequent statistical data analysis based on ARID1B immunoreactivity score were performed to examine the correlation between clinicopathological parameters in breast cancer and ARID1B expression. In-vitro assays were also performed to study the role of ARID1B in cell cycle progression. Univariate analysis revealed that high ARID1B expression is correlated closely with histological grade (P = 0.045) and size (P = 0.043) of invasive breast cancer. These findings were confirmed by multivariate analysis. Notably, increased ARID1B expression was frequently detected in the aggressive triple-negative breast cancer subtypes (P = 0.039) and associated with decreased 5-year disease-free survival rate. Lastly, MDA-MB-231 cells with reduced ARID1B activity displayed a delay in G1 to S phase cell cycle transition and consequently showed a decrease in cell proliferation compared with controls (P < 0.001). ARID1B potentially serves as a valuable prognostic and predictive biomarker as well as a therapeutic target in breast cancer.

Rho-associated protein kinase inhibitor, Y-27632, significantly enhances cell adhesion and induces a delay in G1 to S phase transition in rabbit corneal endothelial cells.

Human corneal endothelial cells are a non-proliferative cell type. As a result of the increase in corneal endothelium disease, increasing numbers of studies have been conducted in order to promote corneal endothelial cell proliferation. The aim of the present study was to investigate the proliferative effects of Rho-associated protein kinase inhibitor, Y-27632, on rabbit corneal endothelial cells (rCECs). Y-27632 (1, 10 or 30 μM) was added at two different time points to two groups of rCECs. The first group received Y-27632 when rCECs were initially plated, and the second following 72 h of cell growth. Cell morphology and cell adhesion ratios were subsequently observed using light microscopy. A cell counting kit was used to measure the number of viable cells that adhered to culture plates. Cell cycle transitions and levels of Annexin V-positive apoptotic cells were detected using flow cytometry. Cells treated with 1 μM Y-27632 and 10 μM Y-27632 retained their cell shape. At a concentration of 30 μM Y-27632, the cell shape became irregular. Cell adhesion ratios, in 1 μM Y-27632 (36.84%), 10 μM Y-27632 (84.21%) and 30 μM Y-27632 (84.21%) were higher than the adhesion ratio in the control group (P<0.01). The optical densities of rCECs treated with 10 μM or 30 μM Y-27632 following 72 h of cell growth was less than that of the control cells (P<0.01), but higher than that of cells which received Y-27632 at the time of plating (P<0.01). Flow cytometry results also demonstrated that there was a delay in G1 to S phase cell cycle progression in rCECs following administration of 10 μM Y-27632 (P<0.01). Cell apoptosis was inhibited when 10 μM Y-27632 was added, at the time of cell plating, as well as when added following 72 h of cell growth (P<0.01). At a concentration of 10 μM Y-27632, there was an improvement in cell adhesion and an inhibition of the cell cycle in rabbit corneal endothelial cells. In conclusion, Y-27632 has different effects on rCECs when administered at varying concentrations and at particular stages of cell growth.