The effect of chemotherapeutic agents on telomere length maintenance in breast cancer cell lines

Copyright @ 2014 the authors. This article is made available through the Brunel Open Access Publishing Fund. It is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability

Mechanisms of Cell Growth

Cell growth, the addition of mass and volume, is required for the development and homeostasis of all organisms. In mammalian cells, cell growth and size homeostasis usually requires an instructive signal in the form of a growth factor with loss of this signal resulting in cell atrophy. The primary Schwann cell has proven a powerful system to study cell growth in vitro – underlining the requirement of growth factors for mammalian cells biogenesis. Using this system, IGF-1 was identified as a Schwann cell growth factor that drives cell volume addition. In contrast, NRG-1 has no effect on Schwann cell volume, but does drive mitochondrial biogenesis - highlighting that cell growth can be non-uniform, diverging from the simple coordinate addition of volume and organelles. In this thesis, I demonstrate that the addition of mass and volume can be uncoupled during cell growth. IGF-1 drives coordinate addition of cell mass and volume, whereas sustained Raf/MEK/ERK activation drives addition of protein mass and specific organelles in the absence of an increase in cell volume. Furthermore, the addition of volume can be uncoupled from mass accumulation, downstream of IGF-1. This demonstrates that factors other than protein mass limit volume addition. To investigate the regulation of cell volume I took two approaches: Firstly, a cellular approach - comparing plasma membrane dynamics in growing and growth factor-starved cells. I show that membrane turnover is extremely fast with the whole membrane internalised ~ three times an hour. Moreover, this rapid and ATP-dependent rate is maintained in starved, autophagic cells - indicating that it is an essential cellular process. The speed of membrane turnover however, precluded using this approach to identify how cells deliver membrane to the cell surface to drive plasma membrane expansion. Secondly, a biochemical approach - to identify important signalling pathways. I identify a critical role for de novo lipogenesis for both the addition and maintenance of cell volume and SREBP-2 as an essential transcription factor mediating this process

Telomere elongation in the breast cancer cell line 21NT after treatment with an epigenetic modifying drug

Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.We thank Professor Robert Newbold for his support and for providing the opportunity to carry out this work within the Institute of Cancer Genetics and Pharmacogenomics, Brunel University London. HY was supported by a triennial project grant (Strategic Award) from the National Centre for Replacement, Refinement, and Reduction (NC3Rs) of animals in research (NC. K500045.1 and G0800697)

Holographic Superconductors from Einstein-Maxwell-Dilaton Gravity

We construct holographic superconductors from Einstein-Maxwell-dilaton gravity in 3+1 dimensions with two adjustable couplings $\alpha$ and the charge $q$ carried by the scalar field. For the values of $\alpha$ and $q$ we consider, there is always a critical temperature at which a second order phase transition occurs between a hairy black hole and the AdS RN black hole in the canonical ensemble, which can be identified with the superconducting phase transition of the dual field theory. We calculate the electric conductivity of the dual superconductor and find that for the values of $\alpha$ and $q$ where $\alpha/q$ is small the dual superconductor has similar properties to the minimal model, while for the values of $\alpha$ and $q$ where $\alpha/q$ is large enough, the electric conductivity of the dual superconductor exhibits novel properties at low frequencies where it shows a "Drude Peak" in the real part of the conductivity.Comment: 25 pages, 13 figures; v2, typos corrected; v3, refs added, to appear in JHE

Thermodynamics of Holographic Defects

Using the AdS/CFT correspondence, we study the thermodynamic properties and the phase diagram of matter fields on (2+1)-dimensional defects coupled to a (3+1)-dimensional N=4 SYM "heat bath". Considering a background magnetic field, (net) quark density, defect "magnitude" $\delta N_c$ and the mass of the matter, we study the defect contribution to the thermodynamic potentials and their first and second derivatives to map the phases and study their physical properties. We find some features that are qualitatively similar to other systems e.g. in (3+1) dimensions and a number of features that are particular to the defect nature, such as its magnetic properties, unexpected properties at T->0 and finite density; and the finite $\delta N_c$ effects, e.g. a diverging susceptibility and vanishing density of states at small temperatures, a physically consistent negative heat capacity and new types of consistent phases.Comment: 33 pages, 16 figures (jpg and pdf), typos fixed and references added, final version published in JHE

Analytic study of properties of holographic p-wave superconductors

In this paper, we analytically investigate the properties of p-wave holographic superconductors in $AdS_{4}$-Schwarzschild background by two approaches, one based on the Sturm-Liouville eigenvalue problem and the other based on the matching of the solutions to the field equations near the horizon and near the asymptotic $AdS$ region. The relation between the critical temperature and the charge density has been obtained and the dependence of the expectation value of the condensation operator on the temperature has been found. Our results are in very good agreement with the existing numerical results. The critical exponent of the condensation also comes out to be 1/2 which is the universal value in the mean field theory.Comment: Latex, To appear in JHE

Low temperature properties of holographic condensates

In the current work we study various models of holographic superconductors at low temperature. Generically the zero temperature limit of those models are solitonic solution with a zero sized horizon. Here we generalized simple version of those zero temperature solutions to small but non-zero temperature T. We confine ourselves to cases where near horizon geometry is AdS^4. At a non-zero temperature a small horizon would form deep inside this AdS^4 which does not disturb the UV physics. The resulting geometry may be matched with the zero temperature solution at an intermediate length scale. We understand this matching from separation of scales by setting up a perturbative expansion in gauge potential. We have a better analytic control in abelian case and quantities may be expressed in terms of hypergeometric function. From this we calculate low temperature behavior of various quatities like entropy, charge density and specific heat etc. We also calculate various energy gaps associated with p-wave holographic superconductor to understand the underlying pairing mechanism. The result deviates significantly from the corresponding weak coupling BCS counterpart.Comment: 17 Page

Rethinking how external pressure can suppress dendrites in lithium metal batteries

We offer an explanation for how dendrite growth can be inhibited when Li metal pouch cells are subjected to external loads, even for cells using soft, thin separators. We develop a contact mechanics model for tracking Li surface and sub-surface stresses where electrodes have realistically (micron-scale) rough surfaces. Existing models examine a single, micron-scale Li metal protrusion under a fixed local current density that presses more or less conformally against a separator or stiff electrolyte. At the larger, sub-mm scales studied here, contact between the Li metal and the separator is heterogeneous and far from conformal for surfaces with realistic roughness: the load is carried at just the tallest asperities, where stresses reach tens of MPa, while most of the Li surface feels no force at all. Yet, dendrite growth is suppressed over the entire Li surface. To explain this dendrite suppression, our electrochemical/mechanics model suggests that Li avoids plating at the tips of growing Li dendrites if there is sufficient local stress; that local contact stresses there may be high enough to close separator pores so that incremental Li+ ions plate elsewhere; and that creep ensures that Li protrusions are gradually flattened. These mechanisms cannot be captured by single-dendrite-scale analyses

Two-week dietary soy supplementation has an estrogenic effect on normal premenopausal breast.

An association has been reported between consumption of a high soy diet and a low incidence of breast cancer within populations of Southeast Asia. Phytoestrogens present in soy act as partial estrogen agonists or antagonists and can inhibit breast cancer cell proliferation in vitro. The effect of 14-day dietary soy supplementation with 60 g (45 mg isoflavones) on the normal breast of 84 premenopausal patients was determined. Serum concentrations of the isoflavanoids, genistein, daidzein, and equol, were raised in patients after soy supplementation (P < or = 0.025). Nipple aspirate (NA) levels of genistein and daidzein were higher than paired serum levels, both before (P < 0.001 and P = 0.001, respectively) and after soy supplementation (P < 0.001 and P = 0.049, respectively); however, there was no significant increase in NA isoflavone levels in response to soy. NA levels of apolipoprotein D were significantly lowered and pS2 levels raised in response to soy supplementation (P < or = 0.002), indicative of an estrogenic stimulus. No effect of soy supplementation on breast epithelial cell proliferation, estrogen and progesterone receptor status, apoptosis, mitosis, or Bcl-2 expression was detected. In conclusion, short term dietary soy has a weak estrogenic response on the breast, as measured by nipple aspirate apolipoprotein D and pS2 expression. No antiestrogenic effect of soy on the breast was detected

Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation

When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal