Molecular mechanisms of drug resistance in a triple negative breast cancer cell model

p53 is a transcription factor activated by genotoxic stress. Dependent on the level of DNA damage, p53 can either trigger cell cycle arrest and DNA repair or programmed cell death. DNA damaging agents that activate p53 are commonly used in the treatment of cancer. If p53 inducible proteins that promote arrest and repair and inhibit apoptosis are up-regulated, tumours can become resistant to many types of treatment (multidrug resistance, MDR). MDR leads to treatment failure, metastases, and death in breast cancer patients. It is especially important in patients who rely solely on chemotherapy because they do not express hormone receptors and thus, do not benefit from endocrine therapies (e.g. tamoxifen). We have generated a triple-negative doxorubicin resistant breast cancer cell line (CALDOX) and shown by RNA array and qPCR analysis that p53-inducible cell survival factor (p53CSV), a p53-inducible inhibitor of apoptosis, is up-regulated in CALDOX and inducible by doxorubicin. Transient knockdowns of p53CSV sensitised CALDOX cells to doxorubicin, but stable knockdowns failed to support this preliminary data. However, CALDOX cells maintained expression of arrest and repair proteins p21 and p53CSV at high levels of genotoxic stress by doxorubicin—while CAL51 cells induced apoptotic protein p53AIP1 and underwent apoptosis. Although it is unlikely that p53CSV acts as a sole factor in MDR in CALDOX cells, p53CSV and other p53 arrest and repair proteins are likely an important factor in MDR through the evasion of apoptosis and promotion of DNA repair in CALDOX cells. Interestingly, we find that p53CSV is widely expressed in normal healthy tissues, conserved across eukaryotes, and in CAL51 cells, only partially p53-dependent—indicating that p53CSV may have an important role separate of p53, perhaps in the control of proliferating cells during development.Imperial Users Onl

Exploring tsRNA Function in Aggressive Breast Cancer Cell Models

Abstract Breast cancer is highly prevalent in the United States with an estimated 260,000 women diagnosed with invasive breast cancer in 2018 alone. There is a growing need to identify the molecular drivers of metastatic breast cancer as the molecular mechanisms responsible for the transition from normal mammary epithelial cells to aggressive cancer cells remain poorly understood. Understanding this transition may reveal a therapeutic target for aggressive breast cancer. Small, noncoding RNAs (ncRNA), such as microRNAs (miRNAs), have recently been discovered to promote initiation, progression, and metastasis of breast cancer. Similar in size to miRNAs, tRNA-derived small RNAs (tsRNAs) are a novel class of small ncRNA whose expression may differentiate between cancer types and cancer cell lines. TsRNAs are created during the maturation process of primary tRNA transcripts, where the 3-prime end of the tRNA is cleaved by RNaseZ, resulting in a 16-48 nucleotide long strand of RNA. Although similar in size to miRNA, the functions of tsRNA are largely unknown. Previously identified two tsRNA, ts-2 and ts-112, that are expressed at 10-fold higher levels in the MCF10CA1a aggressive breast cancer cell line than the normal-like MCF10A mammary epithelial cell line. Further, ts-2 and ts-112 are detected at similarly high levels in female human embryonic cells, displaying oncofetal expression. For these reasons we hypothesize that ts-2 and ts-112 promote breast cancer characteristics. Custom inhibitors of ts-2 and ts-112 were transfected into the aggressive breast cancer cell line MCF10CA1a in vitro. The following phenotypic assays were conducted to determine the function of ts-2 and ts-112 in the MCF10CA1a cell line: proliferation, cell cycle, and wound healing. Following transfection of ts-2 inhibitors, in the proliferation assay showed a 15-20% reduction in growth of aggressive cancer cells. Ts-2 inhibition also saw an increase in population doubling time of 7% from 12 to 14 hours. These results suggest that ts-2 may play a role in cell cycle progression. Following ts-112 inhibition in the aggressive MCF10CA1a breast cancer cell line, cell cycle analysis revealed a statistically significant decrease in the number of cells in G1 phase and an increase in S phase. Using a candidate approach we analyzed the effect of ts-2 and ts-112 inhibition on G1/S phase and S/G2 phase checkpoint markers by qPCR. The inhibition of these tsRNA showed no effect on the chosen genes. Our data are in support for ts-2 and ts-112 having a role in aggressive breast cancer and may be tumor promoting In on-going studies, the capacity of tsRNAs to act as predictive biomarkers of long-term breast cancer risk is being evaluated in serum collected from women at elevated risk. Analyzing ts-2 and ts-112 biological consequences following inhibition furthers our understanding of the molecular mechanisms that are responsible for aggressive breast cancer. Continued study of tsRNA function could produce a model of their role in aggressive breast cancer and thus metastasis. Understanding tsRNA function in metastatic breast cancer in turn could lead to their use as a possible biomarker or therapeutic target

Aggregate Hazes in Exoplanet Atmospheres

Photochemical hazes have been frequently used to interpret exoplanet transmission spectra that show an upward slope towards shorter wavelengths and weak molecular features. While previous studies have only considered spherical haze particles, photochemical hazes composed of hydrocarbon aggregate particles are common throughout the solar system. We use an aerosol microphysics model to investigate the effect of aggregate photochemical haze particles on transmission spectra of warm exoplanets. We find that the wavelength dependence of the optical depth of aggregate particle hazes is flatter than for spheres since aggregates grow to larger radii. As a result, while spherical haze opacity displays a scattering slope towards shorter wavelengths, aggregate haze opacity can be gray in the optical and NIR, similar to those assumed for condensate cloud decks. We further find that haze opacity increases with increasing production rate, decreasing eddy diffusivity, and increasing monomer size, though the magnitude of the latter effect is dependent on production rate and the atmospheric pressure levels probed. We generate synthetic exoplanet transmission spectra to investigate the effect of these hazes on spectral features. For high haze opacity cases, aggregate hazes lead to flat, nearly featureless spectra, while spherical hazes produce sloped spectra with clear spectral features at long wavelengths. Finally, we generate synthetic transmission spectra of GJ 1214b for aggregate and spherical hazes and compare them to space-based observations. We find that aggregate hazes can reproduce the data significantly better than spherical hazes, assuming a production rate limited by delivery of methane to the upper atmosphere.Comment: 17 figures, accepted to Ap

Tracing Sediment in the Subsurface Using Beryllium-7: Green River Basin, KY

As water flows through areas of limestone, karst inevitably develops, creating sinking streams and sinkholes that transport alumino-silicate particles from the surface into the subsurface. Sediment budget studies on short term scales through karst are rare. Thus, a feasibility study using beryllium-7 (7Be), which attaches to soil particles and can be used as a short-term (2-3 month) tracer of the movement of fine-grained sediment in karst was investigated. Sediment samples were collected from a karstic catchment within Green River basin, Kentucky, both on the surface and in the subsurface along a cave stream. Samples were collected prior to and immediately after a recharge event that transported sediment into the subsurface. The before event samples were analyzed for the activity of 7Be to establish a baseline activity for the isotope. The after event samples were collected from the same locations as the pre-event samples were collected and analyzed for 7Be activity. Average 7Be activities measured after the recharge event were as predicted higher than measured 7Be activities before recharge. Our findings indicate that radioisotope 7Be is a viable tracer for sediment dynamics through karst on short-term time scales. Results demonstrate no strong correlation exists between sediment characteristics and 7Be activity, but correspond to the general increasing trending of mean 7Be activity with increasing average grain size and carbonate composition after recharge. This research is important to understanding sediment movement from the Earth’s surface into the subsurface and subsurface sediment dynamics. The potential applications of further studies of 7Be in karst systems and rapid underground sediment dynamics are multi-variable. Future 7Be studies can be applied to trace contaminants as they move through karst systems, to quantify source to sink sediment budget systems, to understand global carbon cycle sinking mechanisms

Assessing Selective Export of Fluorescent SSA4 Reporters

The process of creating proteins from mRNA is necessary for cell viability. Importantly, the proteins that are generated are differentially regulated so that the cell makes specific proteins in response to its environment. For example, mRNA is selectively exported during stress conditions to allow response to this stress. During heat shock at 42°C, in which proteins are denatured, the gene SSA4 encodes a selectively transported protein chaperone that allows refolding of denatured proteins. This study sought to explore the mechanisms of this selective export. To do this, I analyzed the expression of a fluorescent reporter wherein GFP is expressed with the SSA4 promoter, 5’ UTR, and 3’UTR. S. cerevisiae was used as a model system for eukaryotic expression due to ease of observation for mRNA export. Based on the expression of endogenous SSA4, we hypothesized that the samples incubated at 37°C and 42°C should display increased levels of fluorescence relative to unstressed cells. However, only the samples incubated at 37°C where SSA4 is induced, but where selective mRNA export does not occur, demonstrated increased GFP expression. Therefore, we conclude that our reporter is selectively exporting at 37°C but not at 42°C

VAT compliance in the United Kingdom

This study aimed to uncover the factors that influence Value Added Tax (VAT) compliance. Small businesses from the catering and flooring/furnishing trades in the United Kingdom were sent a questionnaire designed to elicit their views on VAT and related issues. Responses were obtained from 359 businesses. Results showed that VAT compliance in small businesses shares a number of similarities with private income tax compliance: Social norms, equity, economic factors and personality are all important in predicting compliance

What the hack is a hacker?

The purpose of this study is to examine the historical tendencies and characteristics of hackers to create a holistic definition of what it means to be a hacker. By analyzing the contents of the “Hacker Classics” list, it was determined that there is not a single, all-encompassing definition of what it means to be a hacker. Although there are common motivations and ideologies shared between many hackers, the definition of “hacker” continues to change as time and technology does

Integrating Network and Intrinsic Changes in GnRH Neuron Control of Ovulation

Infertility affects 15-20% of couples; failure to ovulate is a common cause. Ovulation is triggered when estradiol switches from negative feedback action on the pituitary and hypothalamus to positive feedback, initiating a surge of gonadotropin-releasing hormone (GnRH) secretion that causes a surge of luteinizing hormone (LH) release, which triggers ovulation. Our understanding of the neurobiological changes underlying the switch from negative to positive feedback is incomplete. High levels of estradiol are essential, and in rodents, the LH surge tends to occur at a specific time-of-day. GnRH neurons, however, do not express the estrogen receptor required for feedback, thus estradiol-sensitive afferents likely convey estradiol information to GnRH neurons. We hypothesized that GnRH neurons switch from negative to positive feedback by integrating multiple changes to their synaptic inputs and intrinsic properties. To investigate the neurobiological mechanisms that underlie surge generation, daily GnRH/LH surges can be induced by ovariectomy and estradiol replacement (OVX+E) in rodents. GnRH neuron activity and release are increased in the afternoon (positive feedback) and decreased in the morning (negative feedback). No time-of-day changes are observed in OVX mice that do not receive an estradiol implant. Previous studies using the daily surge model have elucidated multiple GnRH neuron intrinsic and fast-synaptic changes during the switch from negative to positive feedback. It is unclear which if any of these changes are necessary for increasing GnRH firing rate during positive feedback. We hypothesized that changes to GnRH neuron intrinsic properties culminate in an increase in excitability to current steps during positive feedback and a decrease in excitability during negative feedback. To our surprise, changes to GnRH neuron ionic conductances rendered GnRH neurons more excitable during positive feedback relative to all other groups, but changes to ionic conductances between OVX and negative feedback animals had no net effect on GnRH neuron excitability. A mathematical model using a novel application of a rigorous parameter estimation method predicted that multiple, redundant combinations of changes to GnRH intrinsic conductances can produce the firing response in positive feedback. Changes to two interdependent parameters that determine the kinetics of voltage-gated potassium channels accounted for the similar neural responses during negative feedback and in OVX mice. Although enhancing GnRH neuron excitability is expected to increase firing rate during positive feedback, it is unclear if this change is necessary or if the concomitant increase is fast-synaptic transmission is sufficient for increasing GnRH neural activity during positive feedback. To test this, we used dynamic clamp to inject positive feedback, negative feedback, and OVX postsynaptic conductance trains into cells from positive feedback, negative feedback, and OVX mice. Positive feedback conductance trains were more effective in initiating spiking in cells from all three animal models relative to negative feedback and OVX trains. However, the positive feedback train elicited twice the number of action potentials from positive feedback mice relative to those from all other groups. Lastly, we extended our previous work to measure changes to GnRH neuron excitability and GABAergic inputs during the estrous cycle. We demonstrated that GABA postsynaptic current frequency and GnRH neuron excitability are both increased during positive feedback (proestrus) relative to negative feedback (diestrus) and strikingly similar to changes observed in the daily surge model. Collectively, these studies demonstrate that GnRH neurons act to integrate and amplify multiple signals to increase firing rate during the preovulatory surge.PHDMol & Integrtv Physiology PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155290/1/adamsce_1.pd