Nonquiescent Cells Research Articles

Influence of physical factors on logarithmic, quiescent and non-quiescent S. Cerevisiae cells

The influence of environmental conditions on eukaryotic cells, including Saccharomyces cerevisiae, has been the subject of numerous scientific studies. Among the most important physical factors affecting cellular growth are temperature, pH, UV, osmotic pressure, mechanical, gravitational force, and ultrasound. Furthermore, those physical parameters refer to the abiotic factors of an ecosystem. They affect living organisms and can have either stimulating or negative effects on cells. In this respect, quiescent (Q), non-quiescent (NQ), and logarithmic cells (Log) of S. cerevisiae were used as а model system to assess the effect of different physical stresses on cellular survival rate. Obtained results revealed that cells in the G0 state are more resilient to various physical impacts from the environment. At the same time, logarithmic and NQ cells showed from 10 % to 90 % lower resistance depending on the physical factor. Moreover, the complex action of various abiotic factors on a living cell determines its biological path, namely entering different phases of the cell cycle, triggering pr

Quiescent ovarian cancer cells secrete follistatin to induce chemotherapy resistance in surrounding cells in response to chemotherapy.

We recently reported that the transcription factor NFATC4, in response to chemotherapy, drives cellular quiescence to increase OvCa chemoresistance. The goal of this work was to better understand the mechanisms of NFATC4 driven OvCa chemoresistance. We used RNA-seq to identify NFATC4 mediated differential gene expression. CRISPR-Cas9 and FST neutralizing antibody were used to assess impact of loss of FST function on cell proliferation and chemoresistance. ELISA was used to quantify FST induction in patient samples and in vitro in response to chemotherapy. We found that NFATC4 upregulates follistatin (FST) mRNA and protein expression predominantly in quiescent cells and FST is further upregulated following chemotherapy treatment. FST acts in at least a paracrine manner to induce a p-ATF2 dependent quiescent phenotype and chemoresistance in non-quiescent cells. Consistent with this, CRISPR KO of FST in OvCa cells or antibody mediated neutralization of FST sensitizes OvCa cells to chemotherapy treatment. Similarly, CRISPR KO of FST in tumors increased chemotherapy-mediated tumor eradication in an otherwise chemotherapy resistant tumor model. Suggesting a role for FST in chemoresistance in patients, FST protein in the abdominal fluid of OvCa patients significantly increases within 24 hours of chemotherapy exposure. FST levels decline to baseline levels in patients no longer receiving chemotherapy with no evidence of disease. Furthermore, elevated FST expression in patient tumors is correlated with poor progression free, post-progression free, and overall survival. FST is a novel therapeutic target to improve OvCa response to chemotherapy and potentially reduce recurrence rates.

Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations

The aim of the present study was to compare cellular susceptibility and oxidative stress response of S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations to menadione – a well-known inducer of oxidative stress. Three main approaches were used: microbiological – cell survival, molecular – constant field gel electrophoresis for detection of DNA double-strand breaks (DSB), and biochemical – measurement of reactive oxygen species (ROS) levels, oxidized proteins, lipid peroxidation, glutathione, superoxide dismutase (SOD) and catalase on S. cerevisiae haploid strain BY4741. The doses causing 20% (LD20) and 50% (LD50) lethality were calculated. The effect of menadione as a well-known oxidative stress inducer is compared in the log, Q, and NQ cells. Survival data reveal that Q cells are the most susceptible to menadione with LD50 corresponding to 9 µM menadione. On the other hand, dose-dependent DSB induction is found only in Q cells confirming the results shown above. No effect on DSBs levels is observed in log and NQ cells. Further, the oxidative stress response of the cell populations is clarified. Results show significantly higher levels of SOD and ROS in Q cells than in log cells after the treatment with 100 µM menadione. On the other side, higher induction of oxidized proteins, malondialdehyde, and glutathione is observed after menadione treatment of log cells. Our study provides evidence that Saccharomyces cerevisiae quiescent cells are the most susceptible to the menadione action. It might be suggested that the DNA damaging and genotoxic action of menadione in Saccharomyces cerevisiae quiescent cells could be related to ROS production.

Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity

Having unique genetic machinery and a high degree of conservation with higher eukaryotes, the yeast Saccharomyces cerevisiae is recognised as a smart experimental system for studying the modes of chemical toxicity. The present study was undertaken to elucidate the changes in the intracellular redox homeostasis and key macromolecule structure following exposure to Zeocin. Cell populations of logarithmic, quiescent (Q) and non-quiescent (NQ) cells of Saccharomyces cerevisiae BY4741 were used as a model to examine the cytotoxic effect of this radiomimetic. The levels of endogenous ROS, oxidized lipids, carbonylated proteins, and glutathione were analysed after treatment with Zeocin (IC50). An increase in ROS production and respectively increased oxidative stress was detected in all three types of cell populations, with the highest degree being observed in proliferating S. cerevisiae BY4741 cells. The stress response of both proliferating and stationary phase (Q and NQ) cells to Zeocin included an overexpression of glutathione. The quiescent cells also showed very low DNA susceptibility to high Zeocin concentration (100–300 µg/ml), presented as no induced double-strand breaks (DSBs) in the macromolecule. Based on our research it could be concluded that the cellular physiological state is a critical factor determining the resistance to environmental stress with Q cells being the most robust.

Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

Abstract P078: Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer

Abstract Oncogenic KRAS mutation is common in non-small cell lung cancer (NSCLC) and portends poor outcome. Direct KRASG12C inhibitors have clinical activity; however, intrinsic and acquired resistance to these drugs limits their utility. Aurora A Kinase (AURKA), a mitotic cell cycle regulator, has been considered as a key druggable KRAS effector and mediates adaptive resistance to direct KRASG12C inhibitors. We found that AURKA expression correlated with poor outcome of lung cancer patients in caBIG, GEO and TCGA, and its expression was greater in KRAS-mutated NSCLC cells resistant to KRASG12C inhibitor sotorasib (AMG510) compared to normal human tracheobronchial epithelial (NHTBE), lung fibroblast, and KRAS wild-type cells. We have previously demonstrated synergistic antitumor effects for combination AURKA and WEE1 inhibition. Here, we explored a novel combination of AURKA and KRASG12C inhibition in KRAS(G12C)-mutated NSCLC cells intrinsically resistant to the KRASG12C inhibitor, along with a combination of AURKA and WEE1 inhibition in mutant KRAS(G12C) lung cancer cells with acquired resistance to the inhibitor. To overcome resistance to the KRASG12C inhibitor sotorasib, we tested combination treatment with VIC-1911, a newly synthesized selective AURKA inhibitor, with sotorasib in sotorasib-resistant human lung cancer cells. Cooperative screening, Loewe plotting, and clonogenic survival assays were performed to determine synergistic antitumor effects. In addition, we established KRAS(G12C)-mutated human lung cancer cell lines with acquired resistance to sotorasib through the means of escalated incremental dosing or sorting non-quiescent cells after drug exposure. Synergy was further confirmed with cell cycle distribution, phenotypic mitotic catastrophe on confocal microscopy, and induction of apoptosis. Interestingly, cells intrinsically resistant to sotorasib showed profound synergistic suppressive effect on cancer cell survival with addition of VIC-1911 compared to sotorasib-sensitive cells (Loewe synergy scores: NCI-H1792=16.03; HCC44=14.37; NCI-H358=1.48). Further, the combination of AURKA and WEE1 inhibition synergistically induced greater cell death in NCI-H358 lung carcinoma cells harboring acquired resistance to sotorasib, with dramatic induction of Bim. Moreover, the combination of AURKA and WEE1 inhibition resulted in a synergistic tumor control in KRAS/TP53-mutated lung cancer xenograft models. Our findings suggest AURKA activation leads to both intrinsic and acquired resistance to sotorasib in KRAS(G12C)-mutated NSCLC; therefore, addition of AURKA inhibition to sotorasib may be a promising therapeutic approach in NSCLC with intrinsic resistance to direct KRASG12C inhibitors, while the combination of AURKA and WEE1 inhibition merits exploration in acquired resistance to these agents. Citation Format: Jong Woo Lee, Sundong Kim, Cindy Yang, Barbara Burtness. Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P078.

Caloric restriction causes a distinct reorganization of the lipidome in quiescent and non-quiescent cells of budding yeast.

After budding yeast cells cultured in a nutrient-rich liquid medium with 0.2% glucose (under caloric restriction conditions) or 2% glucose (under non-caloric restriction conditions), ferment glucose to ethanol and then consume ethanol, they enter the stationary phase. The process of their chronological aging begins. At that point, the yeast culture starts to accumulate quiescent and non-quiescent cells. Here, we purified the high- and low-density populations of quiescent and non-quiescent cells from the yeast cultures limited in calorie supply or not. We then employed mass spectrometry-based quantitative lipidomics to assess the aging-associated changes in high- and low-density cells’ lipidomes. We found that caloric restriction, a geroprotective dietary intervention, alters the concentrations of many lipid classes through most of the chronological lifespan of the high- and low-density populations of quiescent and non-quiescent cells. Specifically, caloric restriction decreased triacylglycerol, increased free fatty acid, elevated phospholipid and amplified cardiolipin concentrations. Based on these findings, we propose a hypothetical model for a caloric restriction-dependent reorganization of lipid metabolism in budding yeast’s quiescent and non-quiescent cells. We also discovered that caloric restriction creates lipidomic patterns of these cells that differ from those established by two other robust geroprotectors, namely the tor1Δ mutation and lithocholic acid.

Saccharomyces cerevisiae quiescent cells: cadmium resistance and adaptive response

The budding yeast Saccharomyces cerevisiae is a widely used model organism to investigate the changes occurring in the eukaryotic cell and to predict its possible ‘reaction’ to different environmental factors. Recently it was also shown that these microorganisms possess another advantageous ability: to ‘enter’ into quiescent state (G0). Yeast G0 cells have similar physiological characteristics to those of higher eukaryotes making them a better model for toxicology studies. As cadmium could affect severely human health, the main aim of the present study was to use Saccharomyces cerevisiae quiescent cells to investigate the resistance potential and corresponding adaptive response of eukaryotic cells to elevated cadmium concentrations. Both diploid and haploid yeast strains in logarithmic, quiescent and non-quiescent state were exposed to different concentrations of Cd(NO3)2. The half-maximal inhibitory concentration (IC50) for all tested cell types was 100 µmol/L Cd(NO3)2. The deleterious effects of cadmium on intracellular macromolecule structures were analyzed through evaluating the levels of accumulated reactive oxygen species (ROS) and carbonylated proteins. The highest ROS concentration was measured in logarithmic cells: up to 50% versus about 8% in G0 cells. Significant damage in protein molecules was observed in haploid cells where the protein carbonylation reached levels of 25 µmol/mg. Studying the adaptive response to elevated Cd2+ concentrations revealed that quiescent and non-quiescent cells respond with increased expression of key elements from the antioxidant defense system: reduced glutathione, superoxide dismutase (SOD) and catalase. Furthermore, an additional SOD izoenzyme was detected when diploid and haploid cell populations were exposed to Cd2+.

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells

SummaryGenome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Mechanisms that Link Chronological Aging to Cellular Quiescence in Budding Yeast.

After Saccharomyces cerevisiae cells cultured in a medium with glucose consume glucose, the sub-populations of quiescent and non-quiescent cells develop in the budding yeast culture. An age-related chronology of quiescent and non-quiescent yeast cells within this culture is discussed here. We also describe various hallmarks of quiescent and non-quiescent yeast cells. A complex aging-associated program underlies cellular quiescence in budding yeast. This quiescence program includes a cascade of consecutive cellular events orchestrated by an intricate signaling network. We examine here how caloric restriction, a low-calorie diet that extends lifespan and healthspan in yeast and other eukaryotes, influences the cellular quiescence program in S. cerevisiae. One of the main objectives of this review is to stimulate an exploration of the mechanisms that link cellular quiescence to chronological aging of budding yeast. Yeast chronological aging is defined by the length of time during which a yeast cell remains viable after its growth and division are arrested, and it becomes quiescent. We propose a hypothesis on how caloric restriction can slow chronological aging of S. cerevisiae by altering the chronology and properties of quiescent cells. Our hypothesis posits that caloric restriction delays yeast chronological aging by targeting four different processes within quiescent cells.

Low doses of DNA damaging agents extend Saccharomyces cerevisiae chronological lifespan by promoting entry into quiescence

A variety of mild stresses have been shown to extend lifespan in diverse species through hormesis, which is a beneficial response to a stress or toxin that would cause a negative response at a higher exposure. Whether particular stresses induce hormesis can vary with genotype for a given species, and the underlying mechanisms of lifespan extension are only partly understood in most cases. We show that low doses of the DNA damaging or replication stress agents hydroxyurea, methyl methanesulfonate, 4-nitroquinoline 1-oxide, or Zeocin (a phleomycin derivative) lengthened chronological lifespan in Saccharomyces cerevisiae if cells were exposed during growth, but not if they were exposed during stationary phase. Treatment with these agents did not change mitochondrial activity, increase resistance to acetic acid, ethanol, or heat stress, and three of four treatments did not increase resistance to hydrogen peroxide. Stationary phase yeast populations consist of both quiescent and nonquiescent cells, and all four treatments increased the proportion of quiescent cells. Several mutant strains with deletions in genes that influence quiescence prevented Zeocin treatment from extending lifespan and from increasing the proportion of quiescent stationary phase cells. These data indicate that mild DNA damage stress can extend lifespan by promoting quiescence in the absence of mitohormesis or improved general stress responses that have been frequently associated with improved longevity in other cases of hormesis. Further study of the underlying mechanism may yield new insights into quiescence regulation that will be relevant to healthy aging.

Immunophenotypic analysis of cell cycle status in acute myeloid leukaemia: relationship to cytogenetics, genotype and clinical outcome.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P<0·0001), of cycling cells beyond G1 (P<0·0001) and the speed of cycling (P<0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P=0·002) and faster speed of cycling (P=0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P=0·0002), as was a higher relapse rate (P=0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Preferential Ty1 retromobility in mother cells and nonquiescent stationary phase cells is associated with increased concentrations of total Gag or processed Gag and is inhibited by exposure to a high concentration of calcium.

Retrotransposons are abundant mobile DNA elements in eukaryotic genomes that are more active with age in diverse species. Details of the regulation and consequences of retrotransposon activity during aging remain to be determined. Ty1 retromobility in Saccharomyces cerevisiae is more frequent in mother cells compared to daughter cells, and we found that Ty1 was more mobile in nonquiescent compared to quiescent subpopulations of stationary phase cells. This retromobility asymmetry was absent in mutant strains lacking BRP1 that have reduced expression of the essential Pma1p plasma membrane proton pump, lacking the mRNA decay gene LSM1, and in cells exposed to a high concentration of calcium. Mother cells had higher levels of Ty1 Gag protein than daughters. The proportion of protease-processed Gag decreased as cells transitioned to stationary phase, processed Gag was the dominant form in nonquiescent cells, but was virtually absent from quiescent cells. Treatment with calcium reduced total Gag levels and the proportion of processed Gag, particularly in mother cells. We also found that Ty1 reduced the fitness of proliferating but not stationary phase cells. These findings may be relevant to understanding regulation and consequences of retrotransposons during aging in other organisms, due to conserved impacts and regulation of retrotransposons.

Preparation and Analysis of Saccharomyces cerevisiae Quiescent Cells.

Saccharomyces cerevisiae enter quiescence during extended growth in culture (greater than 7days). Here, we describe a method to separate quiescent from non-quiescent cells by density gradient. We also describe approaches for DAPI staining the chromatin of quiescent cells, measuring quiescent cell viability, and extracting RNA from quiescent cells for use in genomics experiments.

BioSpotlight / Citations

BioSpotlight / Citations

Caloric restriction extends yeast chronological lifespan via a mechanism linking cellular aging to cell cycle regulation, maintenance of a quiescent state, entry into a non-quiescent state and survival in the non-quiescent state.

A yeast culture grown in a nutrient-rich medium initially containing 2% glucose is not limited in calorie supply. When yeast cells cultured in this medium consume glucose, they undergo cell cycle arrest at a checkpoint in late G1 and differentiate into quiescent and non-quiescent cell populations. Studies of such differentiation have provided insights into mechanisms of yeast chronological aging under conditions of excessive calorie intake. Caloric restriction is an aging-delaying dietary intervention. Here, we assessed how caloric restriction influences the differentiation of chronologically aging yeast cultures into quiescent and non-quiescent cells, and how it affects their properties. We found that caloric restriction extends yeast chronological lifespan via a mechanism linking cellular aging to cell cycle regulation, maintenance of quiescence, entry into a non-quiescent state and survival in this state. Our findings suggest that caloric restriction delays yeast chronological aging by causing specific changes in the following: 1) a checkpoint in G1 for cell cycle arrest and entry into a quiescent state; 2) a growth phase in which high-density quiescent cells are committed to become low-density quiescent cells; 3) the differentiation of low-density quiescent cells into low-density non-quiescent cells; and 4) the conversion of high-density quiescent cells into high-density non-quiescent cells.

Adaptive Roles of SSY1 and SIR3 During Cycles of Growth and Starvation in Saccharomyces cerevisiae Populations Enriched for Quiescent or Nonquiescent Cells.

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth–starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population’s fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients (ECM21, RSP5, MSN1, SIR4, and IRA2) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1. Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures.

Increased lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae cell populations in early stationary phase

BackgroundProduction of second-generation bioethanol and other bulk chemicals by yeast fermentation requires cells that tolerate inhibitory lignocellulosic compounds at low pH. Saccharomyces cerevisiae displays high plasticity with regard to inhibitor tolerance, and adaptation of cell populations to process conditions is essential for reaching efficient and robust fermentations.ResultsIn this study, we assessed responses of isogenic yeast cell populations in different physiological states to combinations of acetic acid, vanillin and furfural at low pH. We found that cells in early stationary phase (ESP) exhibited significantly increased tolerance compared to cells in logarithmic phase, and had a similar ability to initiate growth in the presence of inhibitors as pre-adapted cells. The ESP cultures consisted of subpopulations with different buoyant cell densities which were isolated with flotation and analysed separately. These so-called quiescent (Q) and non-quiescent (NQ) cells were found to possess similar abilities to initiate growth in the presence of lignocellulosic inhibitors at pH 3.7, and had similar viabilities under static conditions. Therefore, differentiation into Q-cells was not the cause for increased tolerance of ESP cultures. Flow cytometry analysis of cell viability, intracellular pH and reactive oxygen species levels revealed that tolerant cell populations had a characteristic response upon inhibitor perturbations. Growth in the presence of a combination of inhibitors at low pH correlated with pre-cultures having a high frequency of cells with low pHi and low ROS levels. Furthermore, only a subpopulation of ESP cultures was able to tolerate lignocellulosic inhibitors at low pH, while pre-adapted cell populations displayed an almost uniform high tolerance to the adverse condition. This was in stark contrast to cell populations growing exponentially in non-inhibitory medium that were uniformly sensitive to the inhibitors at low pH.ConclusionsESP cultures of S. cerevisiae were found to have high tolerance to lignocellulosic inhibitors at low pH, and were able to initiate growth to the same degree as cells that were pre-adapted to inhibitors at a slightly acidic pH. Carbon starvation may thus be a potential strategy to prepare cell populations for adjacent stressful environments which may be beneficial from a process perspective for fermentation of non-detoxified lignocellulosic substrates at low pH. Furthermore, flow cytometry analysis of pHi and ROS level distributions in ESP cultures revealed responses that were characteristic for populations with high tolerance to lignocellulosic inhibitors. Measurement of population distribution responses as described herein may be applied to predict the outcome of environmental perturbations and thus can function as feedback for process control of yeast fitness during lignocellulosic fermentation.

Abstract P3-03-17: Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas?

Abstract Cancer stem cells are believed to be responsible for radio- and chemoresistance of malignant tumors. In vitro studies demonstrate that ionizing radiation is capable of reprogramming cancer cells from non-stem state into stem state [1, 2]. Moreover, the embryonic stemness cassette was found to be expressed in tumor cells (including breast cancer cells) after they were polyploidized as a result of genotoxic stress [2, 3], thus prompting us to suppose that the polyploid cells and their descendants released by depolyploidization can possess stem cell characteristics. The aim of the current work is to test whether polyploid cells having stem cell features can be also induced in vivo, namely in locally advanced breast carcinomas as a result of neoadjuvant chemotherapy (NAC), assuming that this process is not autonomous, but rather stipulated by the tumor microenvironment. The study population consisted of 30 breast cancer patients of age ranged from 31 to 75 y. o. diagnosed in the Latvian Oncology Center of the Riga East University Hospital between 2013 and 2015. The tissue specimens were collected after the patients' informed consent was obtained in accordance with the Ethics regulations. The clinico-pathologic information about these patients, including Ki-67 index and the status of ER, PR and HER2 receptors, was obtained from the aforementioned clinics. The majority of patients (n = 28) had locally advanced breast cancer, predominantly Stage III disease. Both diagnostic biopsy and operation material, such as primary tumors surgically removed after NAC using standard doses of paclitaxel and doxorubicin, were subjected to DNA content analysis with image cytometry. Ploidy-related parameters, such as DNA index and the percentage of cells exceeding 4.5c (presumably proliferating and polyploid cells), were determined. Immunofluorescence staining was applied to evaluate expression of such markers/factors as proliferation (Ki-67), stemness (SOX2 and NANOG) and invasiveness (CD44). At the time of diagnosis, 14 patients had primary tumors possessing near-triploid clones, and these cases in comparison with 16 other cases comprised of near-euploid clones had 4.5-fold increase of percentages of cells exceeding the ploidy of 4.5c (p &amp;lt; 0.05) and 1.3-fold increase of percentages of cells positive for Ki-67 (p &amp;gt; 0.05). Of 10 cases diagnosed as “triple-negative”, 6 were near-triploid. Among those cases that showed the resistance to NAC (grades 1 and 2 by Miller-Payne histopathologic scoring), 67% were near-triploid. Polyploidization, which in some resistant cases is gained by NAC, was likely to be attributed to near-triploid clones. Notably, polyploid cells were positive for Ki-67, SOX2, NANOG, and CD44. Thus, these non-quiescent polyploid cells can possess the invasiveness and self-renewal features that were also seen in descendants after depolyploidization. Perhaps, reversible polyploidy plays a definite role in gaining the resistance of tumor cells to chemo- and radiotherapy in vivo. 1. Ghisolfi L. et al. PLoS ONE 2012; 7: e43628. 2. Lagadec C. et al. Stem Cells 2012; 30: 833-44. 3. Salmina K. et al. Exp Cell Res 2010; 316: 2099-112. Citation Format: Gerashchenko BI, Salmina K, Eglitis J, Erenpreisa J. Can polyploid tumor cells possessing stem cell features be induced in resistant breast carcinomas? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-17.

Differentiated cytoplasmic granule formation in quiescent and non-quiescent cells upon chronological aging.

Stationary phase cultures represent a complicated cell population comprising at least two different cell types, quiescent (Q) and non-quiescent (NQ) cells. Q and NQ cells have different lifespans and cell physiologies. However, less is known about the organization of cytosolic protein structures in these two cell types. In this study, we examined Q and NQ cells for the formation of several stationary phase-prevalent granule structures including actin bodies, proteasome storage granules, stress granules, P-bodies, the compartment for unconventional protein secretion (CUPS), and Hsp42-associated stationary phase granules (Hsp42-SPGs). Most of these structures preferentially form in NQ cells, except for Hsp42-SPGs, which are enriched in Q cells. When nutrients are provided, NQ cells enter mitosis less efficiently than Q cells, likely due to the time requirement for reorganizing some granule structures. We observed that heat shock-induced misfolded proteins often colocalize to Hsp42-SPGs, and Q cells clear these protein aggregates more efficiently, suggesting that Hsp42-SPGs may play an important role in the stress resistance of Q cells. Finally, we show that the cell fate of NQ cells is largely irreversible even if they are allowed to reenter mitosis. Our results reveal that the formation of different granule structures may represent the early stage of cell type differentiation in yeast stationary phase cultures.