Senescent Fraction Research Articles

Abstract 3444: Identification of a miRNAs signature of radioresistance in a prostate cancer model

Abstract Introduction Radiotherapy (RT) has a prominent role when used as a single modality or in combination with androgen deprivation therapy (ADT) in the management of prostate cancer patients. However, even with currently available high-dose RT protocols, many patients develop recurrence or “biochemical failure” associated with a rise in PSA, and ultimately reduced overall survival. Key regulators of cancer cell behavior, dysregulation of miRNAs expression is associated with both prostate cancer pathogenesis and treatment resistance. This study aimed to identify a miRNAs signature of radioresistance in a prostate cancer model, as a prerequisite for the future development of a novel pre-treatment assay for the identification of radiotherapy prostate cancer patients at risk of biochemical failure. Methods Two models of radioresistance were used : (1) an isogenic radiation resistant model generated through weekly exposure to 2-Gy fractionated ionising radiation of 22Rv1 (primary) prostate cancer (CaP) cells to a cumulative dose of 60Gy. (2) chronically hypoxic 22Rv1 cells (48hrs). miRNA profiling of radioresistant and wild type cells was performed using the Exiqon miRCURY array and overlapping differentially expressed miRs were identified. Results The radiation survival curve of the resulting subline RR22Rv1 was associated with a significant increase in clonogenic survival (1.3 fold increase in survival after 2Gy and 2.2 fold increase after 10Gy), when compared to both parent 22Rv1 and aged-matched controls. The RR22Rv1 cell line is associated with decreased sensitivity to DNA damage (comet assay), enlargement of the S-phase cell population (PI staining) but no evidence for expansion of the senescent cell fraction (β-Gal assay). miRNA profiling identified 105 significantly differentially expressed miRNAs in RR22Rv1, when compared to the parent and age-matched control 22RV1 cells. A total of 12 miRNA were differentially expressed in chronically hypoxic compared to normoxic 22Rv1 cells. Three candidate miRNAs were associated with a radioresistant phenotype across the models: miR200a, miR210 and miR4284. Functional validation of these candidates is currently under way. Conclusion Available in tissues, serum and urine, miRNAs are ideal candidates for the development of novel biological tests. This study identifies a miRNAs signature of radioresistance in prostate cancer cells with potential for the development of a miRNA-based prognostic assay for radiotherapy prostate cancer patients. Citation Format: Niamh McDermott, Armelle Meunier, Christopher Hernandez, Karen J. Bowman, George D. Jones, Laure Marignol. Identification of a miRNAs signature of radioresistance in a prostate cancer model. [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 3444. doi:10.1158/1538-7445.AM2015-3444

Resveratrol, but not dihydroresveratrol, induces premature senescence in primary human fibroblasts

Resveratrol, trans-3,5,4'-trihydroxystilbene, is a polyphenolic compound which has been reported to mimic the gene expression patterns seen in whole animals undergoing dietary restriction. The mechanism of action of resveratrol remains poorly understood, but modulation of both cellular proliferation and apoptosis has been proposed as important routes by which the molecule may exert its effects. This study reports the effects of both resveratrol and dihydroresveratrol (a primary in vivo metabolite) on the proliferative capacity of human primary fibroblasts. No generalised reduction in the growth fraction was observed when fibroblasts derived from three different tissues were treated with resveratrol at concentrations of 10 μm or less. However, concentrations above 25 μm produced a dose-dependent reduction in proliferation. This loss of the growth fraction was paralleled by an increase in the senescent fraction as determined by staining for senescence associated beta galactosidase and dose recovery studies conducted over a 7-day period. Entry into senescence in response to treatment with resveratrol could be blocked by a 30-min preincubation with the p38 MAP kinase inhibitor SB203580. No effects on proliferation were observed when cells were treated with dihydroresveratrol at concentrations of up to 100 μm.

An integrative biological approach to the analysis of tissue culture data: application to the antitumour agent RHPS4

We describe a mathematical model of cell growth and death and explain how it can be used to integrate data from classic tissue culture experiments on antitumour agents and thus aid the identification of their mechanism of action. Experimental data relating to time- and dose-dependent changes in growth rate, cell cycle distribution, plus apoptotic and senescent fractions, are reinterpreted in terms of modulations to kinetic parameters that describe the rates at which cells transit between phenotypic compartments. The mathematical model is analytical, in the sense that the kinetic parameters are calculated from the experimental data directly, without any fitting process. Since the kinetic parameters are much more directly related to potential molecular targets than are the experimentally measured quantities, this approach can provide a more informative picture of the mechanism of action of the antitumour agent under investigation. We demonstrate the potential value of our model by applying it to data from RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate). This agent is a DNA-interactive pentacyclic acridine for which at least three potential mechanisms of antitumour activity have been identified. Firstly RHPS4 is a telomerase inhibitor, secondly it is a telomerase-independent destabiliser of telomeres, and thirdly it is a telomere-independent binder to genomic DNA. Each mechanism can induce a separate, but overlapping, pattern of cellular responses, making the interpretation of tissue culture data very complex. Here we study the time- and dose-dependent effects of RHPS4 on the HCT116 cell line, and develop a five-compartment mathematical model to interpret the data. Application of the model to the data suggests that RHPS4 increases the rate at which cells became senescent state but, rather surprisingly, actually inhibits the rate of cell death. As a control, we also apply the model to data describing the time- and dose-dependent effects of doxorubicin since its mechanism of action is better characterised.

Quantitative assessment of markers for cell senescence

Cellular senescence, the irreversible loss of replicative capacity, might be a tumour suppressor and a contributor to age-related loss of tissue function. The absence of quantitative tests for reliability of candidate markers for senescent cells is a major drawback in cell population studies. Fibroblasts in culture constitute mixed populations of proliferation-competent and senescent cells, with transition between these with increasing population doublings (PD). We estimated senescent fraction in human and mouse fibroblasts with high precision from easily observed growth curves using a dynamic simulation model. We also determined senescent fractions, at various PD (over a wide range of senescent cell frequencies) using candidate senescence markers: Ki67, p21 (CDKN1A), γH2AX, SAHF and Sen-β-Gal either alone or in combination, and compared with those derived from growth curves. This comparison allowed ranking of candidate markers. High rankings were obtained for Sen-β-Gal, SAHFs and the combination of Ki67 negativity with high (>5 per nucleus) γH2A.X foci density in MRC5 fibroblasts. We demonstrate that this latter marker combination, which can easily be performed in paraffin-embedded tissue, gives quantitative senescent cell frequency estimates in mouse embryonic fibroblast cultures and in mouse intestinal sections. The technique presented is a framework for quantitative assessment of markers for senescence.

Forage evaluation, chemical composition, and in vitro digestibility of continuously grazed star grass

This study evaluated the chemical composition and dry matter (DM) digestibility of leaf, stem, and senescent fractions of star grass (Cynodon plectostachyus Pilger) in summer and winter in the south of Brazil. Four 17.6 ha pastures grazed by growing steers in continuous grazing were used. The experiment was divided into two periods: (I) summer: February to June, when five cuts were made; (II) winter: June to October, with six cuts. The forage samples were collected at nine pasture points every 28 days. The DM availability reduced linearly in the summer. In the winter, after September there was a recovery of DM availability. In the summer, there was a linear reduction in leaf proportion and a linear increase in senescent fraction. In the winter, there was a reduction in stem proportion and the senescent fraction increased. Leaf proportion had a quadratic response. The mean crude protein (CP) levels, neutral detergent fiber (NDF), and acid detergent fiber (ADF) during the summer in the leaves were 11.8, 68.6 and 36.1%. The levels were 5.5, 79.3 and 48.8% for stems and the levels were 3.6, 83.0 and 50.2% for the senescent fraction. During the winter, the average levels of CP, NDF and ADF in the leaves were 12.9, 62.2 and 32.7%. The levels were 4.7, 81.2 and 50.1% for stems and 4.1, 84.6 and 54.0% for the senescent fraction. The percentage of indigestible CP in relation to CP was 20% for leaves, 74% for the senescent fraction, and there was variation with time for the stems with levels between 20 and 90%. The DM digestibility of the forage during the summer had a linear reduction and a quadratic behavior during the winter with an average level of 54% in the summer and 42% in the winter.

A cell kinetic analysis of human umbilical vein endothelial cells

Cultures of normal human cells ‘age’ and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43±0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47±0.3% as assessed by senescence associated β-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31±0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

How the Neighborhood Coherence Principle (NCP) Can Give Rise to Tissue Homeostasis: a Cellular Automaton Approach

Gap junctional intercellular communication (GJIC) might provide a key mechanism for tissue homeostasis. Using a three-dimensional cellular automaton, a model was designed to probe the capacity of the particular cell–cell interaction rule known as the neighbourhood coherence principle (NCP) to produce a control mechanism whereby a cell would be told by its neighbors whether or not to enter a division cycle. A numerical experiment using this model showed that a potent feedback control maintaining a local balance between mitoses, cell differentiation, and death emerged from the sequence of cell growth and maturation events. This regulation stemmed from the interplay of the juvenile and the senescent fractions of a tissue. The regulatory loops shows a variable periodicity which depends on the proportion of daughter cells that take the shortcut to re-enter a division cycle immediately. A distinctive tissue patchiness, an intrinsic feature of systems endowed with the NCP interaction rule, constitutes a predictable feature of real cell systems and allows the formulation of empirically testable hypotheses about the role of GJIC in growth regulation.

The nutrition of ruminants grazing native and improved pastures. I. Seasonal variation in the diet selected by grazing sheep and cattle

The chemical composition of the forage on offer on a native pasture dominated by Poa sieberana and Themeda australis, on a similar native pasture dressed with superphosphate and oversown with white clover, and on two Phalaris aquatica (formerly tuberosa)–Trifolium repens pastures dressed with superphosphate and grazed at different stocking rates during summer, was determined at monthly intervals during 1974 and 1975. Samples were also plucked to simulate grazing in 1975, and extrusa were collected from oesophageally fistulated cattle in both years and from fistulated sheep in 1975. Digestibility and nitrogen content of the diet were determined, and in 1974 the extrusa were manually separated into green and senescent fractions. Samples of rumen liquor were also collected through rumen fistulae, and analysed for ammonia, and total and individual volatile fatty acids. The application of superphosphate and white clover, and the replacement of native grasses with Phalaris aquatica, both induced large increases in nutritive value measured in terms of digestibility, protein, macro- and micro-elements in the forage on offer and in the diet selected. Changes in the composition of the forage on offer were attributable to an increase in green material, and to the greater nutritive value of both green and senescent fractions as the extent of pasture improvement increased. Digestibility, nitrogen and mineral content of the diet selected by cattle and sheep increased with pasture improvement, the nutritive value of the diet selected by sheep being greater on all pastures although the difference was greatest on native pasture. The increase in nutritive value of the diet selected by cattle on improved pasture was associated with an increase in the green component of the diet and in the nutritive value of the green material selected.