Kathryn J. Leyva

Growth Kinetics of Extremely Halophilic Archaea (Family Halobacteriaceae) as Revealed by Arrhenius Plots

Members of the family Halobacteriaceae in the domain Archaea are obligate extreme halophiles. They occupy a variety of hypersaline environments, and their cellular biochemistry functions in a nearly saturated salty milieu. Despite extensive study, a detailed analysis of their growth kinetics is missing. To remedy this, Arrhenius plots for 14 type species of the family were generated. These organisms had maximum growth temperatures ranging from 49 to 58 degrees C. Nine of the organisms exhibited a single temperature optimum, while five grew optimally at more than one temperature. Generation times at these optimal temperatures ranged from 1.5 h (Haloterrigena turkmenica) to 3.0 h (Haloarcula vallismortis and Halorubrum saccharovorum). All shared an inflection point at 31 +/- 4 degrees C, and the temperature characteristics for 12 of the 14 type species were nearly parallel. The other two species (Natronomonas pharaonis and Natronorubrum bangense) had significantly different temperature characteristics, suggesting that the physiology of these strains is different. In addition, these data show that the type species for the family Halobacteriaceae share similar growth kinetics and are capable of much faster growth at higher temperatures than those previously reported.

HDAC Inhibitors as Epigenetic Regulators of the Immune System: Impacts on Cancer Therapy and Inflammatory Diseases

Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments.

Zebrafish keratocyte explant cultures as a wound healing model system: differential gene expression & morphological changes support epithelial-mesenchymal transition.

The control of collective cell migration of zebrafish keratocyte sheets in explant culture is of interest for cell migration and epithelial wound healing and depends on the gene expression profile. In a zebrafish genome array, ∼17.5% of the probe sets were differentially expressed greater than two-fold (p≤0.003) between 1 and 7 days of explant culture. Among the differentially expressed genes were a variety of wound healing-related genes and many of the biomarkers for epithelial-mesenchymal transition (EMT), including a switch from keratin and E-cadherin to vimentin and N-cadherin expression and several EMT-related transcription factors were found to be differentially expressed. Supporting evidence for EMT is seen in both morphological change and rearrangement of the actin cytoskeleton and in expression of cadherins during explant culture with a visible disassembly of the cell sheet. TGFβ1 and TNFα expression were analyzed by qPCR at various time points and peak differential expression of both cytokines occurred at 3 days, indicating that the EMT process is ongoing under conditions routinely used in the study of fish keratocyte motility. These data establish that an EMT process is occurring during zebrafish keratocyte explant culture and support the use of this system as a wound healing model.

Collective cell migration of primary zebrafish keratocytes

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 μm/h but over the course of 24h the rate of leading edge advance decreases to ~23 μm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell-cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell-cell interactions determine the role of keratocytes within the migrating sheet.

Epigenetic Regulation of the Biosynthesis & Enzymatic Modification of Heparan Sulfate Proteoglycans: Implications for Tumorigenesis and Cancer Biomarkers

Emerging evidence suggests that the enzymes in the biosynthetic pathway for the synthesis of heparan sulfate moieties of heparan sulfate proteoglycans (HSPGs) are epigenetically regulated at many levels. As the exact composition of the heparan sulfate portion of the resulting HSPG molecules is critical to the broad spectrum of biological processes involved in oncogenesis, the epigenetic regulation of heparan sulfate biosynthesis has far-reaching effects on many cellular activities related to cancer progression. Given the current focus on developing new anti-cancer therapeutics focused on epigenetic targets, it is important to understand the effects that these emerging therapeutics may have on the synthesis of HSPGs as alterations in HSPG composition may have profound and unanticipated effects. As an introduction, this review will briefly summarize the variety of important roles which HSPGs play in a wide-spectrum of cancer-related cellular and physiological functions and then describe the biosynthesis of the heparan sulfate chains of HSPGs, including how alterations observed in cancer cells serve as potential biomarkers. This review will then focus on detailing the multiple levels of epigenetic regulation of the enzymes in the heparan sulfate synthesis pathway with a particular focus on regulation by miRNA and effects of epigenetic therapies on HSPGs. We will also explore the use of lectins to detect differences in heparan sulfate composition and preview their potential diagnostic and prognostic use in the clinic.

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

BackgroundThe BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes.MethodsSW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot.ResultsTreatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor.ConclusionsWhen compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.

Zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Abstract LB-181: Cutaneous SCC may escape immune surveillance by secreting complement factor H

Developing tumors evade elimination by the immune system using a variety of mechanisms. Evidence suggests that cutaneous squamous cell carcinomas (cSCCs) may evade immune system surveillance by secreting complement factor H (CFH), a regulatory protein of the complement system. As part of its normal function, CFH binds to glycosaminoglycans on the surface of host cells where it acts as a cofactor for Factor I mediated C3b cleavage and accelerates the decay of the C3-convertase C3bBb. This mechanism for protecting self-cells also involves shifting the immune response from a pro-inflammatory T-helper-1 cell to an anti-inflammatory regulatory T-cell response. Data presented here suggest that some cSCCs have the ability to upregulate expression of CFH and studies have shown that this could be in response to the pro-inflammatory cytokine interferon-γ. We suggest that, in our cSCC samples, a potential negative-feedback loop exists in which the initial immune response to the developing tumor triggers expression of an immunosuppressive molecule, allowing the cancer to create an environment conducive to evading immune surveillance mechanisms. Note: This abstract was not presented at the meeting. Citation Format: Ellise Loomis, Christopher Pulford, Chandana Uppalapati, Agnes Pascual, McKale Montgomery, Kathryn J. Leyva, Elizabeth E. Hull. Cutaneous SCC may escape immune surveillance by secreting complement factor H [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-181. doi:10.1158/1538-7445.AM2017-LB-181

Abstract 5141: Inhibition of HDAC1 leads to subtype switching in the SW13 adrenal adenocarcinoma line

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA HDAC (histone deacetylase) enzymes are highly expressed in cancer cells and HDAC inhibitors have shown promise in clinical trials for certain patients in multiple types of cancer. In this context, the ability of the SW13 human adrenal adenocarcinoma line to switch between two well-established subtypes in response to HDAC inhibition is of interest. However, the previously characterized inhibitor (trichostatin A) is a toxic, broad-spectrum inhibitor affecting both class I and class II HDACs which have roles in the nucleus and cytosol, respectively. Multiple HDAC inhibitors are screened for their ability induce switching between the SW13 subtypes to identify which HDAC enzyme is necessary for controlling SW13 subtype switching. Results suggest that inhibition of class I HDAC1 increases the rate of conversion between SW13- to SW13+ cells. The effect of HDAC1 inhibition may be enhanced by HDAC3 inhibition as MS-275, an inhibitor specific for HDAC1 but with some specificity for HDAC3, is most effective, particularly when compared to the efficacy of depudecin, an HDAC1 specific inhibitor. After conversion to SW13+, there is increased acetylation on histone H3 but not histone H4 when compared to levels in SW13-, suggesting that the role of acetylation on the H3 tail plays an essential role in the subtype switch. SW13- cells do not express Brg1 or Brm, the mutually exclusive ATPase subunits of the SWI/SNF complex, and do not have an active SWI/SNF complex. Altered activity of this complex and the increased histone H3 acetylation may contribute to the extensive changes in gene expression observed between SW13+ and SW13- subtypes. Citation Format: Jessica M. Lam, Juliane Daggett, Kathryn J. Leyva, Elizabeth E. Hull. Inhibition of HDAC1 leads to subtype switching in the SW13 adrenal adenocarcinoma line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5141. doi:10.1158/1538-7445.AM2014-5141