Figure 4 Vif-induced cellcyclearrest is partially dependent on MOI. Non-synchronized Jurkat cells were infected with NL4-3 e-n-GFP e-f+r+ (A
and B), e-f+r- (C and D), and e-f-r- (E and F) at the indicated MOIs. (A, C, and E) DNA content of GFP + cells was examined by flow cytometry
using DRAQ5 at 24 and 42 hours post-infection as previously described . The percentage of the G2 and G1 populations were modeled using the Watson Pragmatic cellcycle model and the ratio was plotted . All data were represented as mean ± the SD of triplicates. The ns, single (*), double (**), and triple (***) asterisks denote p > 0.05, p < 0.05, p < 0.01, and p < 0.001, respectively, using a one-way analysis of variance (ANOVA) with multiple-comparison tests (Prism, Graph-Pad Software). For each MOI at each time point the G2/G1 ratio for e-f+r+>e-f+r->e-f-r- with p < 0.00001 as analyzed by a one-way ANOVA with multiple-comparison tests. (B, D, and F) The expression of Vif and Vpr increases with increasing MOIs. Lysates were prepared from infected cells at 24 hours post-infection and analyzed for the expression of viral proteins by immunoblotting. The following antibodies were used: mouse anti-p24-capsid (ARRRP) [55,57], rabbit anti-Vpr (a kind gift from B. Sun), mouse anti-Vif (ARRRP) [54-56], and mouse-anti-b-actin (Sigma-Aldrich). Densitometry of the bands was performed using ImageJ (NIH), and the intensity of each band was normalized to b-actin. The fold change of Vif expression is shown under the immunoblots. These data are representative of three experiments.
cellcyclearrest in G2/M-phase and antiviral activity against PRRSV in SJPL cells.
Effect of DIM and SBE-13 on SJPL cellcycle
We have previously shown, using protein profiling, that AppΔapxICΔapxIIC culture supernatant upregulates CDC25c (ser216) and CDK1/2 (tyr161), both of which are implicated in the G2/M cellcycle regulation path- way. Two specific cellcycle inhibitors were used to ver- ify whether cellcycle modulation induced by the culture supernatant is due to CDC25c and/or CDK1/2. We first used DIM which is a specific activator of cellcycle in- hibitor serine/threonine-protein kinase (Chk2), Chk2 is an inhibitor of CDC25 and leads to a cellcyclearrest in G2/M-phase . Then, we used SBE-13 which is a se- lective inhibitor of cellcycle activator polo-kinase iso- forms: PLK1, PLK2, PLK3 . These proteins are implicated in the G2/M-phase transition pathway; PLK1 is a CDC25c activator and CDC25c promotes the G2/
To explore this dichotomy, we investigated the potential that infected cells might also be succumbing to autophagy, another form of active programmed cell death that has been associated with cytotoxicity following stress-induced cellcyclearrest in tumor cells (19, 23, 57). Moreover, p27 accumulation has also been shown to induce autophagy in some tumor cell models in the absence of significant apoptosis (24, 57). Since autophagy is characterized by the formation of autophagic vacuoles that subsequently fuse with lysosomes to form low-pH autolyso- somes (22), infected and uninfected cells were stained with the fluorescent dye Lysotracker Red, and the abundance of acidic vesicles was assessed by flow cytometry. As shown in Fig. 7, no significant differences were observed after 8 h between mock- infected cells and cells infected with either virus. However, at 24 hpi the mean fluorescence detected in cell cultures infected with vMyxT5KO was increased by 3.5-fold compared to mock- infected cells, indicating a greater abundance of lysosomal staining, while the mean fluorescence in vMyxlac-infected cells did not vary significantly from controls (Fig. 7). Moreover, the extent of this effect was greatly reduced when cells were in- fected in the presence of the known inhibitor of autophagy, 3-methyladenine (3-MA) (8). Although mean fluorescence re- mained greater in cultures infected with vMyxT5KO compared to the other two groups, treatment with 3-MA reduced this staining by approximately 50% (Fig. 7). These findings impli- cated M-T5 in the regulation of diverse cell death pathways induced following cellcyclearrest.
showed lower IC 50 values than EG and GA in the majority of the cancer cells.
Interestingly, GA induced cellcyclearrest differentially in PC3, Hep3B and HepG2 cells, where we observed an S phase arrest, than in HeLa cells where we observed a G2/M phase arrest. Previously, it was demonstrated that GA treatment of human prostate carcinoma DU145 cells resulted in an S phase cellcyclearrest [ 20 ]. Recently, it was elucidated that GA induced G2/M phase arrest in HeLa cells accompanied by mitotic catastrophe, and formation of cells with multiple nuclei, followed by impaired centrosomal clustering [ 21 ]. Similarly, TA induced S phase arrest in Hep3B whereas it induced a G2/M phase arrest in HepG2 and PC3 cells. A preceding study in malignant human cholangiocytes indicated for TA an S phase cellcyclearrest [ 22 ]. Finally EG induced a clear G2/M phase cellcyclearrest in the hepatocellular carcinoma Hep3B and HepG2 cells. One explanation of a cellcycle G2/M phase arrest is the inhibition by stabilization or destabilization of microtubules polymerization as that can be observed with taxol or podophillotoxin. Our results showed by confocal microscopy a microtubules stabilization of Hep3B cells treated with EG. Similar results but to a lesser extent were observed with TA. Interestingly we expected to observe an effect on microtubules destabilization rather than stabilization since it has been previously shown that the 3,4,5-trimethoxyphenyl unit of GA, present in the EG structure, is crucial for interactions disturbing the assembly of tubulin [ 23 , 24 ]. Therefore, further studies on the activity of EG on microtubule dynamics in vitro are necessary to better understand the EG mechanism of action.
The present data represent further analysis of previously described rfa1 mutants, rfa1-1, rfa1-9, rfa1-10, rfa1-11 and rfa1-12 . Importantly for the comprehension of the present work, these mutations were shown to be resistant to methylmethane sulfonate treatment, like the wild type but unlike ddc1 and rad51 , and were therefore presumed to be proficient in DNA repair . Here, these mutants have been shown to confer deregulation of the cellcyclearrest that is normally triggered upon telomere uncapping, provoked by the temperature-sensitive cdc13-1 mutation. This should not have been surprising, since a similar phenotype had already been reported concerning the rfa1-t11 mutant (; see also Fig. 1C). Suppression of cdc13-1 arrest by the rfa1-t11 mutation has been shown to be due to an inability for the Rfa1-t11 protein to recruit the Mec1-Ddc2 checkpoint complex . However, suppression of cdc13-1 arrest by the rfa1 mutations analyzed here was nevertheless surprising since these mutants were previously reported to be checkpoint proficient, in this same genetic background , as we confirmed here (Fig. 2A). By genetics, we further demonstrated here that these Rfa1 mutant proteins acted in a separate pathway than that controlled by the Mec1-Rad53-Mec3 DNA damage checkpoint (Fig. 2B). In fact, this was obvious when examining the morphology of the mutant cells of the RPA pathway and comparing with that of the mutant cells of the checkpoint pathway (Fig. 2C). Assessment of telomeric damage strongly suggested that these cdc13-1 rfa1 mutants did not accumulate less damage than the cdc13-1 RFA1 + mutant, which could have explained their improved growth, and did not impinge on the Exo1 pathway of damage generation (Fig. 3).
Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. It works by inhibiting the Tor signaling pathway leading to changes in physiological processes, including cellcyclearrest. In Saccharomyces cerevisiae, rapamycin leads to a rapid and global alteration in gene expression, prompting chromatin remodeling. We propose that histone modification(s) might play a crucial role in remodeling of the chromatin in response to rapamycin. Our main objective is to identify from a histone mutant collection variants that fail to respond to rapamycin in an attempt to characterize histone modifications critical for this drug response. As such, we conducted a screen of the histone mutant collection and identified several hits that showed resistance to rapamycin. We characterized one of the histone variants, namely H2B, carrying alanine substitution at arginine 95 (H2B-R95A) and show that it is extremely resistant to rapamycin, but not to other drugs. Pull downs demonstrated that H2B-R95A was defective in forming a complex with Spt16, an essential factor that is required to disassociate H2A and H2B from the chromatin in order to allow replication and transcription by DNA and RNA polymerases, respectively. ChIP-Chip and microarray experiments showed that arginine 95 of H2B is required to recruit Spt16 to allow expression of several genes, a subset of which are involved in the pheromone signaling pathway. Evidence will be presented to show for the first time that rapamycin can activate the pheromone pathway and that defects in this pathway cause resistance to the drug.
Urine cellcyclearrest biomarkers distinguish poorly between transient and persistent AKI in early septic
shock: a prospective, multicenter study
Dimitri Titeca-Beauport, Delphine Daubin, Ly van Vong, Guillaume Belliard, Cédric Bruel, Sami Alaya, Karim Chaoui, Maud Andrieu, Isabelle
177 Lu-lilotomab activity had to be increased to 500 MBq/kg to show a signi ﬁcant tumor growth delay. Clonogenic and
proliferation assays showed that DOHH2 cells were highly sensitive to 177 Lu-lilotomab, while Ramos cells were the least sensitive, and U2932 (DLBCL), OCI-Ly8, and Rec-1 (mantle cell lymphoma) cells displayed intermediate sensitivity. The strong 177 Lu-lilotomab cytotoxicity observed in DOHH2 cells correlated with reduced G2/M cellcyclearrest, lower WEE-1- and MYT-1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1), and higher apoptosis. In agreement, 177 Lu-lilotomab ef ﬁcacy in vitro, in vivo, and in patient samples was increased when combined with G2/M cellcyclearrest inhibitors (MK-1775 and PD-166285). These results indicate that 177 Lu-lilotomab is particularly ef ﬁcient in treating tumors with reduced inhibitory CDK1 phosphorylation, such as transformed FL.
cell culture experiments clearly demonstrated a cell pro- liferation blockage induced by EMX2 overexpression and a proliferation restart after EMX2 expression arrest (Fig. 3 a, Additional file 1 : Figure S1d), (2) transcriptome analysis suggested that proliferation reversibility is initi- ated but not completely established at day 16 (Fig. 4 a), (3) cellcycle cytometry analysis showed a significant re- duction of the number of cells in S phase following EMX2 overexpression and a return to initial level when EMX2 expression is blocked (Fig. 5 ), and (4) expression profiles at both the RNA and protein levels of selected cellcycle genes also suggested a reversible G1/S cellcyclearrest (Fig. 4 b-c). Reversibility is a key element when considering inhibition of EMX2 as a potential therapeutic strategy. The reversibility of cellcyclearrest by EMX2 seems to be important in the tissue
To decipher the specific contribution of USF1 and p53 proteins to the regulation of cellcycle progression upon genotoxic stress, we generated stable knock-down (KD) cell lines and synchronized them in G1/early S phase. The effectiveness of the shRNAs used to knock down Usf1 and Trp53 was verified (Figure 2, A and B). Levels of Trp53 mRNA were comparable in Usf1 KD and control cells (sh- CT ) and remained unchanged in response to UVB, whereas the level of p53 protein increased only in UVB-irradiated control cells (Figure 2, A and B). The mRNA and protein levels of p21, the p53-dependent effector of the G1/S arrest, remained low in both Usf1 KD and Trp53 KD cells in response to UVB, whereas they increased in control cells. Furthermore, consistent with findings for Usf1 -/- mice, time course experiments showed that there was no delayed UV-induced p53 and p21 up-regulation in Usf1 and Trp53 KD cells (Figure S2A). These findings showed that the KD cell culture models reproduced features of Usf1 -/- mice. To examine S phase progression upon genotoxic stress, we followed the synthesis of DNA by measuring the incorporation of a thymidine analog (BrdU). The results show that the proliferation rates of synchronized Usf1 and Trp53 KD cells were similar to that of control cells (Figure 2C). However, in the UVB-irradiated condition, while the number of BrdU-incorporating cells remained unchanged and comparable for the Usf1 and Trp53 KD cells, irradiated control cells exhibited a significant reduction, of 50%, in the number of BrdU-incorporating cells. Similar results were obtained using primary fibroblasts isolated from Usf1 -/- mice and Usf1 +/+ littermates (Figure S3). These data are consistent with the in vivo results (Figure 1, E and F). In addition, USF1 levels did not differ between Trp53 KD cells and controls, indicating that USF1 expression is not dependent on p53 (Figure 2, A and B). This also suggests that the deficiency in cellcyclearrest of Usf1 KD cells in response to genotoxic stress may be the result of the absence of increased levels and/or activity of p53 and p21.
vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on
noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-in- duced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H 2 O 2 ) accumulation in the
extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glu- tathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A “redoxome” approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cellcyclearrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and bio- logical experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.
There is accumulating evidence that cell-cycle control in mouse embryonic stem (ES) and somatic cells differs in that the mouse ES cell-cycle is not dependent on a functional p16 ink4a /cyclin D:Cyclin-dependent kinase (Cdk)4/pRB:E2F pathway [1-5]. Rather, the cyclin E:Cdk2 and cyclin A:Cdk2 complexes in ES cells are constitutively active throughout the cell-cycle suggesting that the mitotic cycle is constitutively primed for DNA replication . Interestingly, mouse ES cells have a very short G1 phase of approximately 1.5 hr and they predominantly express the hyperphosphorylated form of the retinoblastoma protein (pRB) (specific of the S and G2/M phases of the cell-cycle) , indicating that newly formed cells can enter a new phase of DNA replication very shortly after exit from mitosis. Another striking feature of the mitotic cycle of mouse ES cells is the lack of dependency on serum stimulation  and Mitogen Activated Protein Kinase Kinase (MEK)-associated signalling [4,9,10]. Mouse ES cells rely on Phosphatidyl Inositol-3 kinase (PI3K)-dependent signalling for progression through the G1 phase  as well as for inhibition of differentiation . However, PI3K activity is not dependent on persistent serum stimulation , but rather largely relies both on stimulation of the Leukemia Inhibitory Factor (LIF) receptor and on expression of the ES cell-specific Eras factor . In addition, mouse ES cells do not undergo cell-cyclearrest at the G1 checkpoint in response to DNA damage or nucleotide depletion [13,14], although they synthetize abundant quantities of transcriptionally active p53 . Taken together, these findings indicate that the mitotic cycle of ES cells largely escapes from external mitogenic stimuli and instead relies largely on intrinsic factors.
uncharacterized pathway mediates cellcyclearrest in G2/prophase in response to microtubule perturbations caused by benomyl and low temperature stress. We do not know much about this response, the factors involved, or the nature of the signal triggering it - possible candidates for signals would be unattached kinetochores or the level of free tubulin dimers. It is clear, however, that one of the consequences of this response is a dramatic change in gene expression. How are these changes in transcription mediated? Obvious targets of the response mechanism to microtubule depolymerization would be transcription factors involved in stress response or mitotic and meiotic gene expression. Meiosis is controlled by a complex transcriptional cascade (Kassir et al., 2003). Induction of early meiotic genes is necessary for the correct expression of the subsequent middle and mid-late meiotic genes. Thus, observed delays in induction of later meiotic genes are likely a consequence of a failure early in the expression cascade. However, the transcriptional decrease also coordinately affects general cellcycle factors including most components of the APC/C and other genes involved in chromosome segregation and cellcycle progression. We therefore favor the idea that the expression and/or activity of a number of mitotic and meiotic transcriptional regulators might be coordinately decreased in response to microtubule depolymerization. Indeed the meiotic transcription factors IME1,
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ABSTRACT We studied the steady-state responses to changes in growth rate of yeast when ethanol is the sole source of carbon and energy. Analysis of these data, together with data from studies where glucose was the carbon source, allowed us to distinguish a “universal” growth rate response (GRR) common to all media studied from a GRR specific to the carbon source. Genes with positive universal GRR include ribosomal, translation, and mitochondrial genes, and those with negative GRR include autophagy, vacuolar, and stress response genes. The carbon source–specific GRR genes control mitochondrial function, peroxisomes, and syn- thesis of vitamins and cofactors, suggesting this response may reflect the intensity of oxida- tive metabolism. All genes with universal GRR, which comprise 25% of the genome, are ex- pressed periodically in the yeast metabolic cycle (YMC). We propose that the universal GRR may be accounted for by changes in the relative durations of the YMC phases. This idea is supported by oxygen consumption data from metabolically synchronized cultures with dou- bling times ranging from 5 to 14 h. We found that the high oxygen consumption phase of the YMC can coincide exactly with the S phase of the cell division cycle, suggesting that oxidative metabolism and DNA replication are not incompatible.
CCP-dependent Cdc2 activity may therefore account for the major differences between wild type and MCN cells in the absence of Cdc2 inhibitory phosphorylation. To test this hypothesis, we supplemented our model of MCN cells with a “generic” CCP-dependent Cdc2 activity, re- flecting the situation in cdc13-L-cdc2 Δcdc13 cdc2 + CCP + cells. These additional cyclins account for additional sources of Cdc2 activity throughout the cellcycle, but their temporal patterns are unknown, except for Cig2 [ 20 ]. Therefore, in the extended model, this generic Cdc2 activity is represented simply by a parameter, CCP = 1, and its only effect is to promote the phosphoryla- tion and degradation of Rum1 [ 5 , 21 ]. As a result, the peak of Rum1 accumulation is lowered ( Fig. 4A , left panel) and the range of the stable L steady state is restricted ( Fig. 4A , middle panel) compared to MCN cells ( Fig. 2A ). However, the cellcycle properties of these two strains are nearly identical in the model ( Table 2 Rows 1 and 5), as the cellcycle trajectory remains far from the L steady state. We have confirmed these predictions by examining the phenotype of the corresponding cdc13-L-cdc2 Δcdc13 cdc2 + CCP + strain ( Fig. 4A right panel and Table 1 Row 10). The only discrepancy is that the observed cell size at division is slightly smaller than MCN cells, which could be caused by a small effect of CCPs on mitotic control.
Figure 2. Gene expression profile of the C. albicans cellcycle. Left to right, phaseogram representing transcript level ratios of the 494 cellcycle-regulated genes (y-axis) collected at each of the seven time points (30-min intervals; x-axis). Levels of expression ratios are color coded as follows: yellow (induced), blue (repressed) and gray (no data). The colored bar adjunct to the expression profile reflects our clustering of genes into four major waves of expression (red, G1/S; blue, S/G2; green, G2/M; and orange, M/G1). Representative expression patterns of 30 genes per cluster are illustrated further on the right. The associated table describes for each cluster—the major gene onthology terms, keynote genes, transcription regulatory motifs found, their significance (in C. albicans only and in Candida clade), and the transcription factor known to bind these motifs. In the keynote genes section, cellcycle regulators are underlined and transcription factors are in bold and underlined. A complete list of C. albicans periodically regulated genes can be found in Supplemental Table 1. For p value calculation details, see Materials and Methods. Candida clade includes the following species: C. albicans, C. tropicalis, C. parapsilopsis, D. hansenii, C. guillimondii, L.
In cycling cells, pre-RCs form as cells exit mitosis. Cells enter quiescence from mitosis. The presence of pre-RCs at a subset of origins in G0 implies either that when cells commit to cell-cycle exit pre-RCs only form at some origins or else that pre-RCs form at all origins but are only retained at some. During Drosophila embryogenesis, cells switch at the 16 th division from a cycle lacking G1 to one in which G1 follows mitosis. During this transitionary division, pre-RCs do not form at the exit from mitosis, suggesting that this temporal coupling is not obligatory (Su and O'Farrell, 1997). In contrast, examination of a fluorescently-labeled MCM subunit in S. pombe starved of nitrogen suggests that pre- RCs are first formed coming from a final mitosis and then lost as cells enter G0 (Namdar and Kearsey, 2006). Furthermore, nuclei from human Swiss 3T3 cells loose the ability to replicate in a Xenopus extract slowly, over the course of many days (Sun et al., 2000). This residual replicative capacity is independent of exogenous MCMs, implying a gradual loss of MCMs over time. However in both fission yeast and in human cells pre-RCs are absent from all origins in G0 and it is unclear whether budding yeast regulate pre-RC loss similarly.
in bioelectrical parameters of the cell, such as the transmembrane potential, en- zyme and cofactors charges and the intracellular (pHi). Perturbing one of these parameters has been reported to change the others. So that, cell metabolism seems to be the result of intertwined state parameter oscillations. In this literature investigation, we deciphered cellcycle progression from cell metabolism or more precisely central carbon metabolism (CCM) point of view. It appeared, first, the intriguing relationship between CCM and cellcycle progression, with the reactive and oxidative (redox) cofactors such as NAD + /NADH, NADP + /NADPH being key regulators. Secondly, as reported, mitochondria seem to be more than just a plant for ATP synthesis. They are at the core of eukaryotic cell metabolism and cellcycle progression. In there, the tricarboxylic acid (TCA) cycle, branched to gly- colysis and to the pentose phosphate pathway, is central in mitochondrial metab- olism and has been reported to match mitosis. The TCA is also an adaptive circuit at the crossroads between cytosolic-mitochondrial energy exchanges which are especially enhanced when resting cells are committed to divide. Finally, the progression of the cellcycle exhibits a shifted metabolism, materialized by a shunt from catabolism to anabolism. Transitions are performed by redox potential vari- ation, involving NAD + /NADH, NADP + /NADPH redox couples, and ADP/ATP en- ergetic ratios and the intracellular pH seems to the be master operator of cytosol/ mitochondrial flux balances. Understanding the dynamics of these metabolic ex- changes will pave the way to therapeutic solutions for metabolic cycle disorders such as cancer.