Kirsten H Limesand

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• Limesand, K. H., Nguyen, T., Wong, A., & Chibly, A. (2016). Sensitivity of the salivary glands to radiation: Understanding the inefficient regenerative response and potential therapeutic interventions.. In Salivary Gland Regeneration.
• Morgan-bathke, M., Martin, K., & Limesand, K. H. (2014). Salivary Glands and Saliva. In Encyclopedia of Human Biology. Elsevier Inc. doi:10.1016/b978-0-12-801238-3.00048-9
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  The salivary glands are exocrine organs that secrete the complex fluid of saliva through ducts into the external environment. This article has four critical sections that focus on (1) anatomy of the minor and major salivary glands, (2) cellular architecture of parotid, submandibular, and sublingual glands, (3) regulation of stimulated secretion, and (4) components and functions of saliva. These sections highlight the interdisciplinary nature of salivary research and clinical utility of salivary diagnostics.

Journals/Publications

• Buss, L. G., Rheinheimer, B. A., & Limesand, K. H. (2024). Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands. Scientific reports, 14(1), 845.
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  Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.
• Gunning, J. A., & Limesand, K. H. (2024). Chronic Phenotypes Underlying Radiation-Induced Salivary Gland Dysfunction. Journal of dental research, 103(8), 778-786.
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  Head and neck cancer (HNC) is the sixth most diagnosed cancer, and treatment typically consists of surgical removal of the tumor followed by ionizing radiation (IR). While excellent at controlling tumor growth, IR often damages salivary glands due to their proximity to common tumor sites. Radiation damage to salivary glands results in loss of secretory function, causing severe and chronic reductions in salivary flow. This leads to the patient-reported sensation of dry mouth, termed , which significantly reduces quality of life for HNC patients and survivors. The mechanisms underlying salivary gland damage remain elusive, and therefore, treatment options are scarce. Available therapies provide temporary symptom relief, but there is no standard of care for permanent restoration of function. There is a significant gap in understanding the chronic mechanistic responses to radiation as well as treatments that can be given in the months to years following cessation of treatment. HNC cases are steadily rising; particularly, the number of young patients diagnosed with nonfatal human papillomavirus + HNC continues to increase. The growing number of HNC diagnoses and improved prognoses results in more people living with xerostomia, which highlights the mounting need for restorative treatments. Mechanisms underlying chronic damage include decreases in acinar differentiation markers, increases in acinar cell proliferation, immune and inflammatory dysregulation, and metabolic changes including increases in amino acids and reductions in glycolysis and oxidative phosphorylation, fibrosis, and dysregulated neuronal responses. Currently, promising treatment options include adenoviral gene transfers and stem cell therapy. Thus, this review describes in depth known mechanisms contributing to chronic damage and discusses therapeutic advances in treating chronically damaged glands. Understanding the chronic response to radiation offers potential in development of new therapeutics to reverse salivary gland damage and improve the quality of life of HNC survivors.
• Gunning, J. A., Gilman, K. E., Zúñiga, T. M., Simpson, R. J., & Limesand, K. H. (2024). Parotid glands have a dysregulated immune response following radiation therapy. PloS one, 19(3), e0297387.
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  Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors.
• Buss, L. G., De Oliveira Pessoa, D., Snider, J. M., Padi, M., Martinez, J. A., & Limesand, K. H. (2023). Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages. PLoS ONE, 18(Issue 11). doi:10.1371/journal.pone.0294355
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  Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.
• Buss, L. G., De Oliveira Pessoa, D., Snider, J. M., Padi, M., Martinez, J. A., & Limesand, K. H. (2023). Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages. PloS one, 18(11), e0294355.
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  Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.
• McCabe, S., Limesand, K., & Zhao, N. (2023). Recent progress toward understanding the role of ZIP14 in regulating systemic manganese homeostasis. Computational and Structural Biotechnology Journal, 21(Issue). doi:10.1016/j.csbj.2023.03.039
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  ZIP14 is a metal transporter essential for the regulation of body manganese homeostasis. The physiological functions of ZIP14 have been uncovered mainly through two lines of in vivo studies that examined the phenotypes of ZIP14 loss, including studies of humans with ZIP14 mutations and animals with ZIP14 deficiency. This mini review aims at presenting an updated view of the important advances made towards understanding the genetic and pathological mechanisms of brain manganese overload caused by ZIP14 deficiency.
• Meyer, R. K., Gilman, K. E., Rheinheimer, B. A., Meeks, L., & Limesand, K. H. (2023). AMPK Activation Restores Salivary Function Following Radiation Treatment. Journal of dental research, 102(5), 546-554.
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  Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζ-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.
• Rheinheimer, B., Pasquale, M., Limesand, K., Hoffman, M., & Chibly, A. (2023). Evaluating the transcriptional landscape and cell-cell communication networks in chronically irradiated parotid glands. iScience, 26(5). doi:10.1016/j.isci.2023.106660
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  Understanding the transcriptional landscape that results in chronic salivary hypofunction after irradiation will help identify injury mechanisms and develop regenerative therapies. We present scRNA-seq analysis from control and irradiated murine parotid glands collected 10 months after irradiation. We identify a population of secretory cells defined by specific expression of Etv1, which may be an acinar cell precursor. Acinar and Etv1+ secretory express Ntrk2 and Erbb3, respectively while the ligands for these receptors are expressed in myoepithelial and stromal cells. Furthermore, our data suggests that secretory cells and CD4+CD8+T-cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement. Lastly, evaluation of cell-cell communication networks predicts that neurotrophin, neuregulin, ECM, and immune signaling are dysregulated after irradiation, and thus may play a role in the lack of repair. This resource will be helpful to understand cell-specific pathways that may be targeted to repair chronic damage in irradiated glands.
• Gilman, K. E., & Limesand, K. H. (2021). The complex role of prostaglandin E2-EP receptor signaling in wound healing.. American journal of physiology. Regulatory, integrative and comparative physiology, 320(3), R287-R296. doi:10.1152/ajpregu.00185.2020
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  Prostaglandins are critical lipid mediators involved in the wound healing response, with prostaglandin E2 (PGE2) being the most complex and exhibiting the most diverse physiological outputs. PGE2 signals via four G protein-coupled receptors, termed EP-receptors 1-4 that induce distinct signaling pathways upon activation and lead to an array of different outputs. Recent studies examining the role of PGE2 and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.
• Gilman, K. E., Camden, J. M., Woods, L. T., Weisman, G. A., & Limesand, K. H. (2021). Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E Signaling. Frontiers in bioengineering and biotechnology, 9, 697671.
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  Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E (PGE) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R mice compared to wild-type mice. However, the role of PGE signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE induces salivary gland dysfunction, primary parotid gland cells treated with PGE have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The effects of blocking PGE production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans.
• Meeks, L., De Oliveira Pessoa, D., Martinez, J. A., Limesand, K. H., & Padi, M. (2021). Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction. Physiological genomics, 53(3), 85-98.
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  Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.
• Gilman, K. E., & Limesand, K. H. (2020). The complex role of Prostaglandin E-EP receptor signaling in wound healing. American journal of physiology. Regulatory, integrative and comparative physiology.
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  Prostaglandins are critical lipid mediators involved in the wound healing response, with prostaglandin E (PGE) being the most complex and exhibiting the most diverse physiological outputs. PGE signals via four G-protein coupled receptors, termed EP-receptors 1-4, that induce distinct signaling pathways upon activation and lead to an array of different outputs. Recent studies examining the role of PGE and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.
• Jasmer, K. J., Gilman, K. E., Muñoz Forti, K., Weisman, G. A., & Limesand, K. H. (2020). Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions. Journal of clinical medicine, 9(12).
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  Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.
• Khalafalla, M. G., Woods, L. T., Jasmer, K. J., Forti, K. M., Camden, J. M., Jensen, J. L., Limesand, K. H., Galtung, H. K., & Weisman, G. A. (2020). P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction. Frontiers in pharmacology, 11, 222.
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  Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.
• Mulic, A., Tveit, A., Vieira, N., Limesand, K. H., & Vieira, A. (2020). Protein Profiles of Individuals with Erosive Tooth Wear. Pesquisa Brasileira em Odontopediatria e Clínica Integrada, 20, e0026. doi:https://doi.org/10.1590/pboci.2020.133
• Vieira, A., Vieira, N., Limesand, K. H., & Modesto, A. (2020). Differences in Proteomic Profiles Between Caries Free and Caries Affected Children. Pesquisa Brasileira em Odontopediatria e Clínica Integrada;, 20, e5592. doi:https://doi.org/10.1590/pboci.2020.131
• Winslow, J. W., Limesand, K. H., & Zhao, N. (2020). The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis. International journal of molecular sciences, 21(9).
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  As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters (), (), and () have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.
• Wong, W. Y., Gilman, K., & Limesand, K. H. (2020). Yap activation in irradiated parotid salivary glands is regulated by ROCK activity. PloS one, 15(11), e0232921.
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  Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients' quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.
• Gilman, K. E., Camden, J. M., Klein, R. R., Zhang, Q., Weisman, G. A., & Limesand, K. H. (2019). P2X7 receptor deletion suppresses γ-radiation-induced hyposalivation. American journal of physiology. Regulatory, integrative and comparative physiology, 316(5), R687-R696.
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  Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E (PGE) release from primary parotid cells of wild-type but not P2X7R mice. To attempt to uncover the mechanism underlying differential PGE release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE release in P2X7R mice. Irradiated P2X7R mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.
• Jensen, S. B., Vissink, A., Limesand, K. H., & Reyland, M. E. (2019). Salivary Gland Hypofunction and Xerostomia in Head and Neck Radiation Patients. Journal of the National Cancer Institute. Monographs, 2019(53).
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  The most manifest long-term consequences of radiation therapy in the head and neck cancer patient are salivary gland hypofunction and a sensation of oral dryness (xerostomia).
• Meyer, R., Wong, W. Y., Guzman, R., Burd, R., & Limesand, K. (2019). Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics. Journal of visualized experiments : JoVE.
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  Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.
• Wong, W. Y., Allie, S., & Limesand, K. H. (2019). PKCζ and JNK signaling regulate radiation-induced compensatory proliferation in parotid salivary glands. PloS one, 14(7), e0219572.
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  Radiotherapy is a common treatment option for head and neck cancer patients; however, the surrounding healthy salivary glands are often incidentally irradiated during the process. As a result, patients often experience persistent xerostomia and hyposalivation, which deceases their quality of life. Clinically, there is currently no standard of care available to restore salivary function. Repair of epithelial wounds involves cellular proliferation and establishment of polarity in order to regenerate the tissue. This process is partially mediated by protein kinase C zeta (PKCζ), an apical polarity regulator; however, its role following radiation damage is not completely understood. Using an in vivo radiation model, we show a significant decrease in active PKCζ in irradiated murine parotid glands, which correlates with increased proliferation that is sustained through 30 days post-irradiation. Additionally, salivary glands in PKCζ null mice show increased basal proliferation which radiation treatment did not further potentiate. Radiation damage also activates Jun N-terminal kinase (JNK), a proliferation-inducing mitogen-activated protein kinase normally inhibited by PKCζ. In both a PKCζ null mouse model and in primary salivary gland cell cultures treated with a PKCζ inhibitor, there was increased JNK activity and production of downstream proliferative transcripts. Collectively, these findings provide a potential molecular link by which PKCζ suppression following radiation damage promotes JNK activation and radiation-induced compensatory proliferation in the salivary gland.
• Chibly, A. M., Wong, W. Y., Pier, M., Cheng, H., Mu, Y., Chen, J., Ghosh, S., & Limesand, K. H. (2018). aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1. Scientific reports, 8(1), 6347.
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  Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.
• Lin, H. H., Chung, Y., Cheng, C. T., Ouyang, C., Fu, Y., Kuo, C. Y., Chi, K. K., Sadeghi, M., Chu, P., Kung, H. J., Li, C. F., Limesand, K. H., & Ann, D. K. (2018). Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis. Autophagy, 14(9), 1481-1498.
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  Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene Atg5 (atg5-KO) extends the survival of KRAS-driven tumor-bearing mice by 38%. atg5-KO tumors spread more slowly during late tumorigenesis, despite a faster onset. atg5-KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for de novo asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRAS-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.
• Lin, H. H., Limesand, K. H., & Ann, D. K. (2018). Current State of Knowledge on Salivary Gland Cancers. Critical reviews in oncogenesis, 23(3-4), 139-151.
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  Salivary gland cancers (SGCs), categorized as head and neck cancers (HNCs), constitute about 6% of head and neck cancer diagnoses based on estimate by American Head and Neck Society. Salivary gland tumors originate from different glandular cell types and are thus morphologically diverse. These tumors arise from any of the three major and various minor salivary glands. The incidence of SGCs has slowly increased during the last four decades. The etiology of SGCs is mostly unknown; however, specific gene mutations are associated with certain types of salivary tumors. Treatment options include surgical resection, radiation therapy (RT), chemotherapy, and multimodality therapy. HNC patients treated with RT often develop xerostomia and salivary hypofunction due to damaged salivary glands. In this review, we discuss etiology of SGCs, present findings on the role of autophagy in salivary tumorigenesis, review adverse effects of radiation treatment, and examine remedies for restoration of salivary function.
• Nguyen, V. T., Dawson, P., Zhang, Q., Harris, Z., & Limesand, K. H. (2018). Administration of growth factors promotes salisphere formation from irradiated parotid salivary glands. PloS one, 13(3), e0193942.
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  Worldwide, 500,000 cases of head and neck cancer (HNC) are reported each year and the primary treatment for HNC is radiotherapy. Although the goal of radiotherapy is to target the tumor, secondary exposure occurs in surrounding normal tissues, such as the salivary glands. As a result, despite successful treatment of the cancer, patients are left with long-term side effects due to direct damage to the salivary glands. The effect is chronic and currently there is no treatment. Stem cells are an attractive therapeutic option for treatment of radiation-induced glandular dysfunction because of the potential to regenerate damaged cell populations and restore salivary gland function. However, limited knowledge about the endogenous stem cell population post irradiation hinders the development for stem cell-based therapies. In this study, an ex vivo sphere formation cell culture system was utilized to assess the self-renewal capacity of cells derived from parotid salivary glands at a chronic time point following radiation. Salivary glands from irradiated mice generate significantly fewer salispheres, but can be stimulated with fetal bovine serum (FBS) to generate an equivalent number of salispheres as unirradiated salivary glands. Interestingly, the number and size of salispheres formed is dependent on the concentration of FBS supplemented into the media. Salispheres derived from irradiated glands and cultured in FBS media were found to contain cells that proliferate and express progenitor and acinar cell markers such as Keratin 5, Keratin 14, Aquaporin 5, and NKCC1. Utilization of insulin-like growth factor (IGF1) injections following radiation treatment restores salivary gland function and improves salisphere generation. These findings indicate that stimulation of these cellular populations may provide a promising avenue for the development of cell-based therapies for radiation-induced salivary gland damage.
• Wong, W. Y., Pier, M., & Limesand, K. H. (2018). Persistent disruption of lateral junctional complexes and actin cytoskeleton in parotid salivary glands following radiation treatment. American journal of physiology. Regulatory, integrative and comparative physiology, 315(4), R656-R667.
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  Xerostomia and hyposalivation are debilitating side effects for patients treated with ionizing radiation for head and neck cancer. Despite technological advances, collateral damage to the salivary glands remains a significant problem for patients and severely diminishes their quality of life. During the wound healing process, restoration of junctional contacts is necessary to maintain polarity, structural integrity, and orientation cues for secretion. However, little is known about whether these structural molecules are impacted following radiation damage and more importantly, during tissue restoration. We evaluated changes in adherens junctions and cytoskeletal regulators in an injury model where mice were irradiated with 5 Gy and a restoration model where mice injected postradiation with insulin-like growth factor 1 (IGF1) are capable of restoring salivary function. Using coimmunoprecipitation, there is a decrease in epithelial (E)-cadherin bound to β-catenin following damage that is restored to untreated levels with IGF1. Via its adaptor proteins, β-catenin links the cadherins to the cytoskeleton and part of this regulation is mediated through Rho-associated coiled-coil containing kinase (ROCK) signaling. In our radiation model, filamentous (F)-actin organization is fragmented, and there is an induction of ROCK activity. However, a ROCK inhibitor, Y-27632, prevents E-cadherin/β-catenin dissociation following radiation treatment. These findings illustrate that radiation induces a ROCK-dependent disruption of the cadherin-catenin complex and alters F-actin organization at stages of damage when hyposalivation is observed. Understanding the regulation of these components will be critical in the discovery of therapeutics that have the potential to restore function in polarized epithelium.
• Khalafalla, M. G., Woods, L. T., Camden, J. M., Khan, A. A., Limesand, K. H., Petris, M. J., Erb, L., & Weisman, G. A. (2017). P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy. The Journal of biological chemistry, 292(40), 16626-16637.
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  Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.
• Meyer, S., Chibly, A. M., Burd, R., & Limesand, K. H. (2017). Insulin-Like Growth Factor-1-Mediated DNA Repair in Irradiated Salivary Glands Is Sirtuin-1 Dependent. Journal of dental research, 96(2), 225-232.
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  Ionizing radiation is one of the most common cancer treatments; however, the treatment leads to a wide range of debilitating side effects. In patients with head and neck cancer (HNC), the surrounding normal salivary gland is extremely sensitive to therapeutic radiation, and damage to this tissue results in various oral complications and decreased quality of life (QOL). In the current study, mice treated with targeted head and neck radiation showed a significant increase in double-stranded breaks (DSB) in the DNA of parotid salivary gland cells immediately after treatment, and this remained elevated 3 h posttreatment. In contrast, mice pretreated with insulin-like growth factor-1 (IGF-1) showed resolution of the same amount of initial DNA damage by 3 h posttreatment. At acute time points (30 min to 2 h), irradiated parotid glands had significantly decreased levels of the histone deactylase Sirtuin-1 (SirT-1) which has been previously shown to function in DNA repair. Pretreatment with IGF-1 increased SirT-1 protein levels and increased deacetylation of SirT-1 targets involved in DNA repair. Pharmacological inhibition of SirT-1 activity decreased the IGF-1-mediated resolution of DSB. These data suggest that IGF-1 promotes DNA repair in irradiated parotid glands through the maintenance and activation of SirT-1.
• Burd, R., Chibly, A., Limesand, K., & Meyer, S. (2016). Insulin-Like Growth Factor-1–Mediated DNA Repair in Irradiated Salivary Glands Is Sirtuin-1 Dependent. Journal of Dental Research, 96(2), 225-232. doi:10.1177/0022034516677529
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  Ionizing radiation is one of the most common cancer treatments; however, the treatment leads to a wide range of debilitating side effects. In patients with head and neck cancer (HNC), the surrounding normal salivary gland is extremely sensitive to therapeutic radiation, and damage to this tissue results in various oral complications and decreased quality of life (QOL). In the current study, mice treated with targeted head and neck radiation showed a significant increase in double-stranded breaks (DSB) in the DNA of parotid salivary gland cells immediately after treatment, and this remained elevated 3 h posttreatment. In contrast, mice pretreated with insulin-like growth factor-1 (IGF-1) showed resolution of the same amount of initial DNA damage by 3 h posttreatment. At acute time points (30 min to 2 h), irradiated parotid glands had significantly decreased levels of the histone deactylase Sirtuin-1 (SirT-1) which has been previously shown to function in DNA repair. Pretreatment with IGF-1 increased SirT-1 protein levels and increased deacetylation of SirT-1 targets involved in DNA repair. Pharmacological inhibition of SirT-1 activity decreased the IGF-1–mediated resolution of DSB. These data suggest that IGF-1 promotes DNA repair in irradiated parotid glands through the maintenance and activation of SirT-1.
• Donovan, M. G., Branco, G. M., Burd, R., Harris, Z., & Limesand, K. H. (2016). Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy.. Frontiers in nutrition, 3, 48. doi:10.3389/fnut.2016.00048
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  Replacing current refractory treatments for melanoma with new prevention and therapeutic approaches is crucial in order to successfully treat this aggressive cancer form. Melanoma develops from neural crest cells, which express tyrosinase - a key enzyme in the pigmentation pathway. The tyrosinase enzyme is highly active in melanoma cells and metabolizes polyphenolic compounds; tyrosinase expression thus makes feasible a target for polyphenol-based therapies. For example, quercetin (3,3',4',5,7-pentahydroxyflavone) is a highly ubiquitous and well-classified dietary polyphenol found in various fruits, vegetables, and other plant products including onions, broccoli, kale, oranges, blueberries, apples, and tea. Quercetin has demonstrated antiproliferative and proapoptotic activity in various cancer cell types. Quercetin is readily metabolized by tyrosinase into various compounds that promote anticancer activity; additionally, given that tyrosinase expression increases during tumorigenesis, and its activity is associated with pigmentation changes in both early- and late-stage melanocytic lesions, it suggests that quercetin can be used to target melanoma. In this review, we explore the potential of quercetin as an anti-melanoma agent utilizing and extrapolating on evidence from previous in vitro studies in various human malignant cell lines and propose a "four-focus area strategy" to develop quercetin as a targeted anti-melanoma compound for use as either a preventative or therapeutic agent. The four areas of focus include utilizing quercetin to (i) modulate cellular bioreduction potential and associated signaling cascades, (ii) affect transcription of relevant genes, (iii) regulate epigenetic processes, and (iv) develop effective combination therapies and delivery modalities/protocols. In general, quercetin could be used to exploit tyrosinase activity to prevent, and/or treat, melanoma with minimal additional side effects.
• Goldberg, E. L., Romero-Aleshire, M. J., Renkema, K. R., Ventevogel, M. S., Chew, W. M., Uhrlaub, J. L., Smithey, M. J., Limesand, K. H., Sempowski, G. D., Brooks, H. L., & Nikolich-Žugich, J. (2015). Lifespan-extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms. Aging cell, 14(1), 130-8.
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  Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5-10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.
• Morgan-Bathke, M., Lin, H. H., Ann, D. K., & Limesand, K. H. (2015). The Role of Autophagy in Salivary Gland Homeostasis and Stress Responses. Journal of dental research, 94(8), 1035-40.
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  Autophagy is a catabolic process that has been shown to have a role in many cellular processes including the removal of excessive or damaged proteins and protein aggregates. The salivary glands play a critical role in oral health, and their secretory capacity may be critically intertwined with the autophagic process. This review describes the role of autophagy activation in normal salivary gland homeostasis and during the glandular stress responses of therapeutic radiation, ductal ligation, autoimmunity, and salivary gland adenoid cystic carcinoma.
• Ann, D. K., Burd, R., Chibly, A. M., Harris, Z. I., Hill, G. A., Klein, R. R., Limesand, K. H., Lin, H. H., & Morgan-bathke, M. (2014). Autophagy correlates with maintenance of salivary gland function following radiation.. Scientific reports, 4(1), 5206. doi:10.1038/srep05206
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  The current standard of care for head and neck cancer includes surgical resection of the tumor followed by targeted head and neck radiation. This radiotherapy results in a multitude of negative side effects in adjacent normal tissues. Autophagy is a cellular mechanism that could be targeted to ameliorate these side effects based on its role in cellular homeostasis. In this study, we utilized Atg5(f/f);Aqp5-Cre mice which harbor a conditional knockout of Atg5, in salivary acinar cells. These autophagy-deficient mice display increased radiosensitivity. Treatment of wild-type mice with radiation did not robustly induce autophagy following radiotherapy, however, using a model of preserved salivary gland function by IGF-1-treatment prior to irradiation, we demonstrate increased autophagosome formation 6-8 hours following radiation. Additionally, administration of IGF-1 to Atg5(f/f);Aqp5-Cre mice did not preserve physiological function. Thus, autophagy appears to play a beneficial role in salivary glands following radiation and pharmacological induction of autophagy could alleviate the negative side effects associated with therapy for head and neck cancer.
• Brooks, H. L., Chew, W. M., Ghosh, S., Goldberg, E. L., Hapak, S. M., Kusne, Y., Limesand, K. H., Maskaykina, I. Y., Nikolich-zugich, J., Parker, S. S., Price, T. J., & Sanai, N. (2014). Contrasting effects of chronic, systemic treatment with mTOR inhibitors rapamycin and metformin on adult neural progenitors in mice.. Age (Dordrecht, Netherlands), 36(1), 199-212. doi:10.1007/s11357-013-9572-5
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  The chronic and systemic administration of rapamycin extends life span in mammals. Rapamycin is a pharmacological inhibitor of mTOR. Metformin also inhibits mTOR signaling but by activating the upstream kinase AMPK. Here we report the effects of chronic and systemic administration of the two mTOR inhibitors, rapamycin and metformin, on adult neural stem cells of the subventricular region and the dendate gyrus of the mouse hippocampus. While rapamycin decreased the number of neural progenitors, metformin-mediated inhibition of mTOR had no such effect. Adult-born neurons are considered important for cognitive and behavioral health, and may contribute to improved health span. Our results demonstrate that distinct approaches of inhibiting mTOR signaling can have significantly different effects on organ function. These results underscore the importance of screening individual mTOR inhibitors on different organs and physiological processes for potential adverse effects that may compromise health span.
• Brooks, H. L., Chew, W. M., Goldberg, E. L., Limesand, K. H., Nikolich‐Žugich, J., Renkema, K. R., Romero‐Aleshire, M. J., Sempowski, G. D., Smithey, M. J., Uhrlaub, J. L., & Ventevogel, M. S. (2014). Lifespan‐extending caloric restriction or m TOR inhibition impair adaptive immunity of old mice by distinct mechanisms. Aging Cell, 14(1), 130-138. doi:10.1111/acel.12280
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  Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5-10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.
• Chibly, A. M., Limesand, K. H., & Nguyen, T. (2014). Palliative Care for Salivary Gland Dysfunction Highlights the Need for Regenerative Therapies: A Review on Radiation and Salivary Gland Stem Cells.. Journal of palliative care & medicine, 4(4). doi:10.4172/2165-7386.1000180
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  Radiotherapy remains the major course of treatment for Head and Neck cancer patients. A common consequence of radiation treatment is dysfunction of the salivary glands, which leads to a number of oral complications including xerostomia and dysphagia, for which there is no existent cure. Here, we briefly describe the current palliative treatments available for patients undergoing these conditions, such as oral lubricants, saliva substitutes, and saliva stimulants. None of these options achieves restoration of normal quality of life due to their limited effectiveness, and in some cases, adverse side effects of their own. Other therapies under development, such as acupuncture and electrostimulation have also yielded mixed results in clinical trials. Due to the ineffectiveness of palliative care to restore quality of life, it is reasonable to aim for the development of regenerative therapies that allow restoration of function of the salivary epithelium following radiation treatment. Adult stem cells are a necessary component of wound healing, and play important roles in preserving normal function of adult tissues. Thus, the present review mainly focuses on the effects of radiation on adult stem cells in a variety of tissues, which may be at play in the response of salivary glands to radiation treatment. This is of clinical importance because progenitor cells of the salivary glands have shown partial regenerative potential in mouse transplantation assays. Therefore, understanding how these progenitor cells are affected by radiation offers potential for development of new therapies for patients with xerostomia.
• Chibly, A., Querin, L., Harris, Z., & Limesand, K. (2014). Label-retaining cells in the adult murine salivary glands possess characteristics of adult progenitor cells. PLoS ONE, 9(9). doi:10.1371/journal.pone.0107893
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  Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.
• Hill, G., Headon, D., Harris, Z. I., Huttner, K., & Limesand, K. H. (2014). Pharmacological activation of the EDA/EDAR signaling pathway restores salivary gland function following radiation-induced damage. PloS one, 9(11), e112840.
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  Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage.
• Lin, H. H., Lin, S. M., Chung, Y., Vonderfecht, S., Camden, J. M., Flodby, P., Borok, Z., Limesand, K. H., Mizushima, N., & Ann, D. K. (2014). Dynamic involvement of ATG5 in cellular stress responses. Cell death & disease, 5, e1478.
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  Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated β-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.
• Morgan-Bathke, M., Harris, Z. I., Arnett, D. G., Klein, R. R., Burd, R., Ann, D. K., & Limesand, K. H. (2014). The Rapalogue, CCI-779, improves salivary gland function following radiation. PloS one, 9(12), e113183.
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  The standard of care for head and neck cancer typically includes surgical resection of the tumor followed by targeted head and neck radiation. However depending on tumor location and stage, some cases may not require surgical resection while others may be treated with chemoradiation. Unfortunately, these radiation treatments cause chronic negative side effects for patients. These side effects are associated with damage to surrounding normal salivary gland tissue and include xerostomia, changes in taste and malnutrition. The underlying mechanisms of chronic radiation-induced salivary gland dysfunction are unknown, however, in rodent models persistently elevated proliferation is correlated with reduced stimulated salivary flow. The rapalogue, CCI-779, has been used in other cell systems to induce autophagy and reduce proliferation, therefore the aim of this study was to determine if CCI-779 could be utilized to ameliorate chronic radiation-induced salivary gland dysfunction. Four to six week old Atg5f/f; Aqp5-Cre, Atg5+/+; Aqp5-Cre and FVB mice were treated with targeted head and neck radiation. FVB mice were treated with CCI-779, chloroquine, or DMSO post-radiation. Stimulated salivary flow rates were determined and parotid and submandibular salivary gland tissues were collected for analyses. Mice with a defect in autophagy, via a conditional knockout of Atg5 in the salivary glands, display increased compensatory proliferation in the acinar cell compartment and hypertrophy at 24-72 hours following radiation. FVB mice treated with post-therapy CCI-779 have significant improvements in salivary gland physiology as determined by stimulated salivary flow rates, proliferation indices and amylase production and secretion. Consequently, post-radiation use of CCI-779 allows for improvement of salivary gland function and reestablishment of glandular homeostasis. As CCI-779 is already FDA approved for other uses, it could have a secondary use to alleviate the chronic side effects in head and neck cancer patients who have completed anti-tumor therapy.
• Ann, D. K., Arnett, D. G., Borok, Z., Chen, C. H., Chibly, A. M., Flodby, P., Klein, R. R., Limesand, K. H., Lin, H. H., Morgan-bathke, M., Sun, X., Wu, R., & Zhang, W. (2013). Deletion of ATG5 shows a role of autophagy in salivary homeostatic control.. Journal of dental research, 92(10), 911-7. doi:10.1177/0022034513499350
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  Autophagy is a catabolic pathway utilized to maintain a balance among the synthesis, degradation, and recycling of cellular components, thereby playing a role in cell growth, development, and homeostasis. Previous studies revealed that a conditional knockout of essential member(s) of autophagy in a variety of tissues causes changes in structure and function of these tissues. Acinar cell-specific expression of knocked-in Cre recombinase through control of aquaporin 5 (Aqp5) promoter/enhancer (Aqp5-Cre) allows us to specifically inactivate Atg5, a protein necessary for autophagy, in salivary acinar cells of Atg5(f/f);Aqp5-Cre mice. There was no difference in apoptotic or proliferation levels in salivary glands of Atg5/Cre mice from each genotype. However, H&E staining and electron microscopy studies revealed modestly enlarged acinar cells and accumulated secretory granules in salivary glands of Atg5(f/f);Aqp5-Cre mice. Salivary flow rates and amylase contents of Atg5/Cre mice indicated that acinar-specific inactivation of ATG5 did not alter carbachol-evoked saliva and amylase secretion. Conversely, autophagy intersected with salivary morphological and secretory manifestations induced by isoproterenol administration. These results identified a role for autophagy as a homeostasis control in salivary glands. Collectively, Atg5(f/f);Aqp5-Cre mice would be a useful tool to enhance our understanding of autophagy in adaptive responses following targeted head and neck radiation or Sjögren syndrome.
• Chibly, A. M., Fribley, A. M., & Limesand, K. H. (2013). Impact of targeting insulin-like growth factor signaling in head and neck cancers.. Growth hormone & IGF research : official journal of the Growth Hormone Research Society and the International IGF Research Society, 23(5), 135-40. doi:10.1016/j.ghir.2013.06.001
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  The IGF system has been shown to have either negative or negligible impact on clinical outcomes of tumor development depending on specific tumor sites or stages. This review focuses on the clinical impact of IGF signaling in head and neck cancer, the effects of IGF targeted therapies, and the multi-dimensional role of IRS 1/2 signaling as a potential mechanism in resistance to targeted therapies. Similar to other tumor sites, both negative and positive correlations between levels of IGF-1/IGF-1-R and clinical outcomes in head and neck cancer have been reported. In addition, utilization of IGF targeted therapies has not demonstrated significant clinical benefit; therefore the prognostic impact of the IGF system on head and neck cancer remains uncertain.
• Arnett, D. G., Burd, R., Hill, G. A., Klein, R. R., Limesand, K. H., & Martin, K. L. (2012). Prevention of radiation-induced salivary gland dysfunction utilizing a CDK inhibitor in a mouse model.. PloS one, 7(12), e51363. doi:10.1371/journal.pone.0051363
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  Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues..Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G(2)/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G(2)/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output..These studies suggest that induction of transient G(2)/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.
• Burd, R., Caro, J., Kong, X., Leeper, D. B., Limesand, K. H., Mendoza, E. E., & Pocceschi, M. G. (2012). Control of Glycolytic Flux by AMP-Activated Protein Kinase in Tumor Cells Adapted to Low pH.. Translational oncology, 5(3), 208-16. doi:10.1593/tlo.11319
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  Tumor cells grow in nutrient- and oxygen-deprived microenvironments and adapt to the suboptimal growth conditions by altering their metabolic pathways. This adaptation process commonly results in a tumor phenotype that displays a high rate of aerobic glycolysis and aggressive tumor characteristics. The glucose regulatory molecule, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), is a bifunctional enzyme that is central to glycolytic flux and is downstream of the metabolic stress sensor AMP-activated protein kinase (AMPK), which has been suggested to modulate glycolysis and possibly activate isoforms of PFKFB, specifically PFKFB3 expressed in tumor cells. Our results demonstrated that long-term low pH exposure induced AMPK activation, which resulted in the up-regulation of PFKFB3 and an increase in its serine residue phosphorylation. Pharmacologic activation of AMPK resulted in an increase in PFKFB3 as well as an increase in glucose consumption, whereas in contrast, inhibition of AMPK resulted in the down-regulation of PFKFB3 and decreased glycolysis. PFKFB3 overexpression in DB-1 tumor cells induced a high rate of glycolysis and inhibited oxygen consumption, confirming its role in controlling glycolytic flux. These results show that low pH is a physiological stress that can promote a glycolytic phenotype commonly associated with tumorigenesis. The implications are that the tumor microenviroment contributes to tumor growth and treatment resistance.
• Arnett, D. G., Burd, R., Fribley, A. M., Klein, R. R., Limesand, K. H., Sittadjody, S., & Victory, K. R. (2011). Head and neck tumor cell radiation response occurs in the presence of IGF1.. Journal of dental research, 90(3), 347-52. doi:10.1177/0022034510388037
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  Radiation therapy for head and neck cancer results in severe secondary side-effects in salivary glands. We previously demonstrated that the administration of IGF1 preserves or restores salivary gland function following radiation. Based on these findings, we propose to study the effect of IGF1 on human head and neck carcinoma cells. Head and neck tumor cells treated with radiation have significant reductions in tumor cell survival, as measured by MTT and crystal violet assays, regardless of IGF1 pre-treatment. Head and neck squamous carcinoma cell xenografts treated with concurrent radiation+IGF1 also exhibit significant tumor growth delay; however, growth rates are elevated compared with those in irradiated xenografts. In contrast, administration of IGF1 after radiation treatment has no effect on tumor xenograft growth rates. Analysis of these data suggests that localized delivery may be required for concurrent therapy to prevent secondary side-effects of radiotherapy, while post-therapy administration of IGF1 could be considered for the restoration of salivary function.
• Burd, R., Limesand, K. H., Mendoza, E. E., Sittadjody, S., Thangasamy, T., & Vargas, A. J. (2011). Exploiting tyrosinase expression and activity in melanocytic tumors: quercetin and the central role of p53.. Integrative cancer therapies, 10(4), 328-40. doi:10.1177/1534735410391661
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  Melanoma is an aggressive tumor that expresses the pigmentation enzyme tyrosinase. Tyrosinase expression increases during tumorigenesis, which could allow for selective treatment of this tumor type by strategies that use tyrosinase activity. Approaches targeting tyrosinase would involve gene transcription or signal transduction pathways mediated by p53 in a direct or indirect manner. Two pathways are proposed for exploiting tyrosinase expression: (a) a p53-dependent pathway leading to apoptosis or arrest and (b) a reactive oxygen species-mediated induction of endoplasmic reticulum stress in p53 mutant tumors. Both strategies could use tyrosinase-mediated activation of quercetin, a dietary polyphenol that induces the expression of p53 and modulates reactive oxygen species. In addition to antitumor signaling properties, activation of quercetin could complement conventional cancer therapy by the induction of phase II detoxification enzymes resulting in p53 stabilization and transduction of its downstream targets. In conclusion, recent advances in tyrosinase enzymology, prodrug chemistry, and modern chemotherapeutics present an intriguing and selective multitherapy targeting system where dietary bioflavonoids could be used to complement conventional cancer treatments.
• Avila, J. L., Burd, R., Fillinger, J. L., Limesand, K. H., Mitchell, G. C., & Sittadjody, S. (2010). IGF1 activates cell cycle arrest following irradiation by reducing binding of ΔNp63 to the p21 promoter.. Cell death & disease, 1(6), e50. doi:10.1038/cddis.2010.28
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  Radiotherapy for head and neck tumors often results in persistent loss of function in salivary glands. Patients suffering from impaired salivary function frequently terminate treatment prematurely because of reduced quality of life caused by malnutrition and other debilitating side-effects. It has been previously shown in mice expressing a constitutively active form of Akt (myr-Akt1), or in mice pretreated with IGF1, apoptosis is suppressed, which correlates with maintained salivary gland function measured by stimulated salivary flow. Induction of cell cycle arrest may be important for this protection by allowing cells time for DNA repair. We have observed increased accumulation of cells in G2/M at acute time-points after irradiation in parotid glands of mice receiving pretreatment with IGF1. As p21, a transcriptional target of the p53 family, is necessary for maintaining G2/M arrest, we analyzed the roles of p53 and p63 in modulating IGF1-stimulated p21 expression. Pretreatment with IGF1 reduces binding of ΔNp63 to the p21 promoter after irradiation, which coincides with increased p53 binding and sustained p21 transcription. Our data indicate a role for ΔNp63 in modulating p53-dependent gene expression and influencing whether a cell death or cell cycle arrest program is initiated.
• Avila, J. L., Chang, H., Grundmann, O., Klein, R. R., Limesand, K. H., Shin, Y. J., & Victory, K. R. (2010). Insulin-like growth factor-1 preserves salivary gland function after fractionated radiation.. International journal of radiation oncology, biology, physics, 78(2), 579-86. doi:10.1016/j.ijrobp.2010.03.035
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  Radiotherapy for head-and-neck cancer consists of fractionated radiation treatments that cause significant damage to salivary glands leading to chronic salivary gland dysfunction with only limited prevention and treatment options currently available. This study examines the feasibility of IGF-1 in preserving salivary gland function following a fractionated radiation treatment regimen in a pre-clinical model..Mice were exposed to fractionated radiation, and salivary gland function and histological analyses of structure, apoptosis, and proliferation were evaluated..In this study, we report that treatment with fractionated doses of radiation results in a significant level of apoptotic cells in FVB mice after each fraction, which is significantly decreased in transgenic mice expressing a constitutively active mutant of Akt1 (myr-Akt1). Salivary gland function is significantly reduced in FVB mice exposed to fractionated radiation; however, myr-Akt1 transgenic mice maintain salivary function under the same treatment conditions. Injection into FVB mice of recombinant insulin-like growth factor-1 (IGF-1), which activates endogenous Akt, suppressed acute apoptosis and preserved salivary gland function after fractionated doses of radiation 30 to 90 days after treatment. FVB mice exposed to fractionated radiation had significantly lower levels of proliferating cell nuclear antigen-positive salivary acinar cells 90 days after treatment, which correlated with a chronic loss of function. In contrast, FVB mice injected with IGF-1 before each radiation treatment exhibited acinar cell proliferation rates similar to those of untreated controls..These studies suggest that activation of IGF-1-mediated pathways before head-and-neck radiation could modulate radiation-induced salivary gland dysfunction and maintain glandular homeostasis.
• Avila, J. L., Fillinger, J. L., Limesand, K. H., Mitchell, G. C., & Victory, K. R. (2010). Binding of {Delta}Np63 to the promoter of p21, a p53-dependent cell cycle arrest gene, modifies the response of salivary glands to therapeutic radiation. The FASEB Journal, 24(S1). doi:10.1096/fasebj.24.1_supplement.930.2
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  Radiotherapy targeting head and neck tumors often results in persistent loss of function in the salivary glands, leading to severe malnutrition other debilitating side effects in these patients. Radiation-induced salivary gland damage results from attrition of salivary cells to p53-dependent apoptosis. In p53 knockout mice as well as in mice expressing a constitutively active form of Akt (myr-Akt1), this apoptosis is suppressed, and slaivary gland function is maintained. Injections of IGF1, a potent activator of Akt in salivary glands, also results in preservation of irradiated glands. Induction of cell cycle arrest may be important for this protection by allowing cells time for extensive DNA repair. We have observed increased accumulation of cells in G2/M and a reduction in proliferation at acute time-points following irradiation in salivary glands of mice receiving pre-treatment with IGF1. IGF1 also leads to a decreased stress response in irradiated glands as indicated by reduced stress-induced phosphorylation of p53 (Ser18) and ΔNp63 (Ser160/162). The ΔN isoforms of p63 are thought to act as dominant negatives to p53-induced gene expression. Given that several p53 target genes, including p21, are involved in cell cycle regulation, this suggests a mechanism by which IGF1’s affects on p63 could modulate arrest. Interestingly, pre-treatment with IGF1 reduces binding of ΔN to the p21 promoter following irradiation, which may allow p53 to transactivate that gene and initiate arrest and repair, resulting in long-term preservation of salivary gland function. Support: NIDCR K22 (DE16096), CBIO T32 (CA09213), NSF GK-12 (0338247).
• Burd, R., Fillinger, J. L., Grundmann, O., Limesand, K. H., & Victory, K. R. (2010). Restoration of Radiation Therapy-induced Salivary Gland Dysfunction in Mice by Post Therapy IGF-1 Administration. International Journal of Radiation Oncology Biology Physics, 78(3), S579. doi:10.1016/j.ijrobp.2010.07.1350
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  Radiotherapy for head and neck cancer results in severe and chronic salivary gland dysfunction in most individuals. This results in significant side effects including xerostomia, dysphagia, and malnutrition which are linked to significant reductions in patients' quality of life. Currently there are few xerostomia treatment approaches that provide long-term results without significant side effects. To address this problem we investigated the potential for post-therapeutic IGF-1 to reverse radiation-induced salivary gland function.
• Burd, R., Fillinger, J. L., Grundmann, O., Limesand, K. H., & Victory, K. R. (2010). Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration.. BMC cancer, 10(1), 417. doi:10.1186/1471-2407-10-417
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  Radiotherapy for head and neck cancer results in severe and chronic salivary gland dysfunction in most individuals. This results in significant side effects including xerostomia, dysphagia, and malnutrition which are linked to significant reductions in patients' quality of life. Currently there are few xerostomia treatment approaches that provide long-term results without significant side effects. To address this problem we investigated the potential for post-therapeutic IGF-1 to reverse radiation-induced salivary gland dysfunction..FVB mice were treated with targeted head and neck radiation and significant reductions in salivary function were confirmed 3 days after treatment. On days 4-8 after radiation, one group of mice was injected intravenously with IGF-1 while a second group served as a vehicle control. Stimulated salivary flow rates were evaluated on days 30, 60, and 90 and histological analysis was performed on days 9, 30, 60, and 90..Irradiated animals receiving vehicle injections have 40-50% reductions in stimulated salivary flow rates throughout the entire time course. Mice receiving injections of IGF-1 have improved stimulated salivary flow rates 30 days after treatment. By days 60-90, IGF-1 injected mice have restored salivary flow rates to unirradiated control mice levels. Parotid tissue sections were stained for amylase as an indicator of functioning acinar cells and significant reductions in total amylase area are detected in irradiated animals compared to unirradiated groups on all days. Post-therapeutic injections of IGF-1 results in increased amylase-positive acinar cell area and improved amylase secretion. Irradiated mice receiving IGF-1 show similar proliferation indices as untreated mice suggesting a return to tissue homeostasis..Post-therapeutic IGF-1 treatment restores salivary gland function potentially through normalization of cell proliferation and improved expression of amylase. These findings could aid in the rational design of therapy protocols or drugs for the treatment of radiation-induced salivary gland dysfunction in patients who have completed their anti-cancer therapies.
• Burd, R., Limesand, K. H., Mendoza, E. E., Mitchell, G. C., Radhakrishnan, V. M., Sittadjody, S., & Thangasamy, T. (2010). Quercetin abrogates chemoresistance in melanoma cells by modulating deltaNp73.. BMC cancer, 10(1), 282. doi:10.1186/1471-2407-10-282
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  The alkylating agent dacarbazine (DTIC) has been used in the treatment of melanoma for decades, but when used as a monotherapy for cancer only moderate response rates are achieved. Recently, the clinical use of temozolomide (TMZ) has become the more commonly used analog of DTIC-related oral agents because of its greater bioavailability and ability to cross the blood brain barrier. The response rates achieved by TMZ are also unsatisfactory, so there is great interest in identifying compounds that could be used in combination therapy. We have previously demonstrated that the bioflavonoid quercetin (Qct) promoted a p53-mediated response and sensitized melanoma to DTIC. Here we demonstrate that Qct also sensitizes cells to TMZ and propose a mechanism that involves the modulation of a truncated p53 family member, deltaNp73..DB-1 melanoma (p53 wildtype), and SK Mel 28 (p53 mutant) cell lines were treated with TMZ (400 microM) for 48 hrs followed by Qct (75 microM) for 24 hrs. Cell death was determined by Annexin V-FITC staining and immunocytochemical analysis was carried out to determine protein translocation..After treatment with TMZ, DB-1 cells demonstrated increased phosphorylation of ataxia telangiectasia mutated (ATM) and p53. However, the cells were resistant to TMZ-induced apoptosis and the resistance was associated with an increase in nuclear localization of deltaNp73. Qct treatment in combination with TMZ abolished drug insensitivity and caused a more than additive induction of apoptosis compared to either treatment alone. Treatment with Qct, caused redistribution of deltaNp73 into the cytoplasm and nucleus, which has been associated with increased p53 transcriptional activity. Knockdown of deltaNp73 restored PARP cleavage in the TMZ treated cells, confirming its anti-apoptotic role. The response to treatment was predominantly p53 mediated as the p53 mutant SK Mel 28 cells showed no significant enhancement of apoptosis..This study demonstrates that Qct can sensitize cells to TMZ and that the mechanisms of sensitization involve modulation of p53 family members.
• Fillinger, J. L., Grundmann, O., Limesand, K. H., & Victory, K. R. (2010). Restoration of radiation therapy-induced salivary gland dysfunction by post therapy IGF1 administration. The FASEB Journal, 24(S1). doi:10.1096/fasebj.24.1_supplement.930.4
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  Radiotherapy for head and neck cancer results in severe and chronic salivary gland dysfunction resulting in significant side effects including xerostomia and malnutrition. To address this problem we investigated the effect of IGF1 on the radiation response in normal tissue. Female mice were treated with targeted head and neck radiation and significant reductions in salivary function were confirmed 3 days after treatment. On days 4–8 after radiation, mice were divided into two groups with one group injected intravenously with IGF1 while the second group served as a vehicle injected control. Irradiated animals have 40–50% reductions in stimulated salivary flow rates. Mice receiving injections of IGF1 have increases in stimulated salivary flow rates 60–90 salivary flow rates that are not significantly different from unirradiated mice. Parotid tissue sections from irradiated animals show significant reductions in total amylase area when compared to unirradiated mice. Post-therapeutic injections of IGF1 results in increased amylase-positive acinar cell area on days 30–90. Post-therapeutic IGF1 treatment restores salivary gland function through normalization of cell proliferation and improved expression of amylase. These findings could aid in the treatment of radiation-induced salivary gland dysfunction in patients who have completed their anti-cancer therapies. Supported in part by DE16096 and DE18888.
• Limesand, K. H., & Victory, K. R. (2010). IGF1 prevents radiation-induced salivary gland dysfunction which does not affect survival of head and neck tumors. The FASEB Journal, 24(S1). doi:10.1096/fasebj.24.1_supplement.930.3
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  Radiation therapy for head and neck cancer results in severe secondary side effects in non-tumor tissue which leads to reduced quality of life for patients. One of the most common secondary side effects is xerostomia, a syndrome that results from salivary glands not producing sufficient quantities of saliva. We developed a preclinical in vivo model where intravenous injection of insulin-like growth factor 1 (IGF-1) stimulated activation of the endogenous Akt in salivary glands. Treatment with IGF-1 in FVB mice prior to ionizing radiation of the head and neck region preserved salivary function. To further understand the affect of IGF1 on tumor clearance in human head and neck squamous cell carcinoma (SCC), we assessed levels of cell survival following gamma radiation. Treatment of human SCC with IGF1 showed no significant increase in tumor cells surviving after gamma radiation, as measured by both MTT and crystal violet assays. These studies suggest that IGF1 preserves salivary function in vivo and does not affect tumor cell proliferation or survival in vitro following radiotherapy. Currently we are investigating the effects on IGF1 in mice with SCC tumor xenographs. Targeting the IGF1 pathway may provide a treatment option that alleviates the secondary side effects of radiotherapy and improves the quality of life for head and neck cancer patients. Support: NIDCR RO1 (18888)
• Reyland, M. E., Baum, B. J., Coppes, R. P., Elting, L. S., Fox, P. C., Jensen, S. B., Langendijk, J. A., Limesand, K. H., Mitchell, J. B., & Vissink, A. (2010). Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers.. International journal of radiation oncology, biology, physics, 78(4), 983-91. doi:10.1016/j.ijrobp.2010.06.052
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  The most significant long-term complication of radiotherapy in the head-and-neck region is hyposalivation and its related complaints, particularily xerostomia. This review addresses the pathophysiology underlying irradiation damage to salivary gland tissue, the consequences of radiation injury, and issues contributing to the clinical management of salivary gland hypofunction and xerostomia. These include ways to (1) prevent or minimize radiation injury of salivary gland tissue, (2) manage radiation-induced hyposalivation and xerostomia, and (3) restore the function of salivary gland tissue damaged by radiotherapy.
• Anderson, S. M., Limesand, K. H., & Said, S. (2009). Suppression of radiation-induced salivary gland dysfunction by IGF-1.. PloS one, 4(3), e4663. doi:10.1371/journal.pone.0004663
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  Radiation is a primary or secondary therapeutic modality for treatment of head and neck cancer. A common side effect of irradiation to the neck and neck region is xerostomia caused by salivary gland dysfunction. Approximately 40,000 new cases of xerostomia result from radiation treatment in the United States each year. The ensuing salivary gland hypofunction results in significant morbidity and diminishes the effectiveness of anti-cancer therapies as well as the quality of life for these patients. Previous studies in a rat model have shown no correlation between induction of apoptosis in the salivary gland and either the immediate or chronic decrease in salivary function following gamma-radiation treatment..A significant level of apoptosis can be detected in the salivary glands of FVB mice following gamma-radiation treatment of the head and neck and this apoptosis is suppressed in transgenic mice expressing an activated mutant of Akt (myr-Akt1). Importantly, this suppression of apoptosis in myr-Akt1 mice preserves salivary function, as measured by saliva output, three and thirty days after gamma-radiation treatment. In order to translate these studies into a preclinal model we found that intravenous injection of IGF1 stimulated activation of endogenous Akt in the salivary glands in vivo. A single injection of IGF1 prior to exposure to gamma-radiation diminishes salivary acinar cell apoptosis and completely preserves salivary gland function three and thirty days following irradiation..These studies suggest that apoptosis of salivary acinar cells underlies salivary gland hypofunction occurring secondary to radiation of the head and neck region. Targeted delivery of IGF1 to the salivary gland of patients receiving head and neck irradiation may be useful in reducing or eliminating xerostomia and restoring quality of life to these patients.
• Avila, J. L., Burd, R., Grundmann, O., & Limesand, K. H. (2009). Radiation-induced salivary gland dysfunction results from p53-dependent apoptosis.. International journal of radiation oncology, biology, physics, 73(2), 523-9. doi:10.1016/j.ijrobp.2008.09.036
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  Radiotherapy for head-and-neck cancer causes adverse secondary side effects in the salivary glands and results in diminished quality of life for the patient. A previous in vivo study in parotid salivary glands demonstrated that targeted head-and-neck irradiation resulted in marked increases in phosphorylated p53 (serine(18)) and apoptosis, which was suppressed in transgenic mice expressing a constitutively active mutant of Akt1 (myr-Akt1)..Transgenic and knockout mouse models were exposed to irradiation, and p53-mediated transcription, apoptosis, and salivary gland dysfunction were analyzed..The proapoptotic p53 target genes PUMA and Bax were induced in parotid salivary glands of mice at early time points after therapeutic radiation. This dose-dependent induction requires expression of p53 because no radiation-induced expression of PUMA and Bax was observed in p53-/- mice. Radiation also induced apoptosis in the parotid gland in a dose-dependent manner, which was p53 dependent. Furthermore, expression of p53 was required for the acute and chronic loss of salivary function after irradiation. In contrast, apoptosis was not induced in p53-/- mice, and their salivary function was preserved after radiation exposure..Apoptosis in the salivary glands after therapeutic head-and-neck irradiation is mediated by p53 and corresponds to salivary gland dysfunction in vivo.
• Burd, R., Limesand, K. H., Sittadjody, S., & Thangasamy, T. (2009). Abstract #5521: Induction of p53 dependent and independent death pathways by targeting cell specific redox systems. Cancer Research, 69, 5521-5521.
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  Background: Glutathione (GSH) and thioredoxin (Trx) play a major role in cellular redox balance and prevent oxidative damage in cells and tissues. In tumors the level of GSH and Trx can be elevated and prevent cell death or apoptosis following chemotherapy or radiation therapy. Hydroxyethyl-disulfide (HEDS) and lipoate (LA) are disulfides that are primarily bioreduced by the GSH and Trx pathways respectively, and can be used to selectively downregulate these redox systems. Quercetin (Qct), an abundant bioflavonoid has been extensively studied because of its redox modulating activity, as well as its ability to inhibit the activity of enzymes involved in signal transduction. Although known as an antioxidant, we have recently shown that high concentrations of quercetin can interfere with disulfide metabolism and can generate ROS. Here we specifically deplete the major bioreduction pathways of different cancer cell lines and demonstrate that intracellular signaling mechanisms can be manipulated to induce cell death via p53-dependent and -independent mechanisms. Methods: A549 lung cancer and U87 glioma cells were treated with HEDS and/or LA in the presence or absence Quercetin (75 µM). Protein phosphorylation was determined by Western blot analysis and densitometry. Cell death and ROS was determined by Annexin V-FITC and DCFH-DA staining, respectively. Results: A549 cells demonstrated a greater capacity to bioreduce LA, whereas U87 cells showed a greater capacity to bioreduce HEDS. Correspondingly, increased levels of ROS, cell detachment and apoptosis were observed by the combination of Qct plus LA in A549 cells and by Qct plus HEDS in U87 cells. Cell death in both cells lines was associated with reduced levels of phospho-Src and phospho-FAK and inhibition of phosphorylation was greater in the presence of Qct, which has been shown to inhibit protein kinase activities. In U87 cells, phospho-FAK suppressed p53 phosphorylation but depletion of GSH by treatment with HEDS inhibited FAK and induced phosphorylation of p53. Apoptosis was induced in both cell lines and was mediated by p53-dependent (caspase-9) and p53-independent (caspase-8) pathways in the U87 and A549 cells, respectively. Conclusion: These results demonstrate that redox levels can be specifically manipulated in the presence of kinase inhibitors to affect the phosphorylation or activation of proteins and enhance cell death. Additionally, knowledge of the cross-talk between cell redox systems and cell signaling or survival pathways will aid in the rational design of small molecule inhibitors. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5521.
• Grundmann, O., Limesand, K. H., & Mitchell, G. C. (2009). Sensitivity of salivary glands to radiation: from animal models to therapies.. Journal of dental research, 88(10), 894-903. doi:10.1177/0022034509343143
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  Radiation therapy for head and neck cancer causes significant secondary side-effects in normal salivary glands, resulting in diminished quality of life for these individuals. Salivary glands are exquisitely sensitive to radiation and display acute and chronic responses to radiotherapy. This review will discuss clinical implications of radiosensitivity in normal salivary glands, compare animal models used to investigate radiation-induced salivary gland damage, address therapeutic advances, and project future directions in the field.
• Burd, R., H. Limesand, K., Sittadjody, S., & Thangasamy, T. (2008). Tyrosinase Overexpression Promotes ATM-Dependent p53 Phosphorylation by Quercetin and Sensitizes Melanoma Cells to Dacarbazine. Analytical Cellular Pathology, 30(5), 371-387. doi:10.1155/2008/764086
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  Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr + cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μ M). Both pcDNA3 and Tyr + DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr + cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr + cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase.
• Burd, R., Limesand, K. H., Sittadjody, S., & Thangasamy, T. (2008). Tyrosinase overexpression promotes ATM-dependent p53 phosphorylation by quercetin and sensitizes melanoma cells to dacarbazine.. Cellular oncology : the official journal of the International Society for Cellular Oncology, 30(5), 371-87. doi:10.3233/clo-2008-0441
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  Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr(+) cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 microM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 microM). Both pcDNA3 and Tyr(+) DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr(+) cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr(+) cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase.
• Thangasamy, T., Sittadjody, S., H. Limesand, K., & Burd, R. (2008). Tyrosinase overexpression promotes ATM-dependent p53 phosphorylation by quercetin and sensitizes melanoma cells to dacarbazine. Cellular Oncology, 30(5). doi:10.3233/CLO-2008-0441
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  Dacarbazine (DTIC) has been used for the treatment of melanoma for decades. However, monotherapy with this chemotherapeutic agent results only in moderate response rates. To improve tumor response to DTIC current clinical trials in melanoma focus on combining a novel targeted agent with chemotherapy. Here, we demonstrate that tyrosinase which is commonly overexpressed in melanoma activates the bioflavonoid quercetin (Qct) and promotes an ataxia telangiectasia mutated (ATM)-dependent DNA damage response. This response sensitizes melanoma cells that overexpress tyrosinase to DTIC. In DB-1 melanoma cells that overexpress tyrosinase (Tyr+ cells), the threshold for phosphorylation of ATM and p53 at serine 15 was observed at a low dose of Qct (25 μM) when compared to the mock transfected pcDNA3 cells, which required a higher dose (75 μM). Both pcDNA3 and Tyr+ DB-1 cells demonstrated similar increases in phosphorylation of p53 at other serine sites, but in the Tyr+ cells, DNApk expression was found to be reduced compared to control cells, indicating a shift towards an ATM-mediated response. The DB-1 control cells were resistant to DTIC, but were sensitized to apoptosis with high dose Qct, while Tyr+ cells were sensitized to DTIC with low or high dose Qct. Qct also sensitized SK Mel 5 (p53 wildtype) and 28 (p53 mutant) cells to DTIC. However, when SK Mel 5 cells were transiently transfected with tyrosinase and treated with Qct plus DTIC, SK Mel 5 cells demonstrated a more than additive induction of apoptosis. Therefore, this study demonstrates that tyrosinase overexpression promotes an ATM-dependent p53 phosphorylation by Qct treatment and sensitizes melanoma cells to dacarbazine. In conclusion, these results suggest that Qct or Qct analogues may significantly improve DTIC response rates in tumors that express tyrosinase. © 2008 - IOS Press and the authors. All rights reserved.
• Burd, R., Lanza-jacoby, S., Limesand, K. H., Sittadjody, S., Thangasamy, T., & Wachsberger, P. R. (2007). Quercetin selectively inhibits bioreduction and enhances apoptosis in melanoma cells that overexpress tyrosinase.. Nutrition and cancer, 59(2), 258-68. doi:10.1080/01635580701499545
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  Tyrosinase is expressed in melanoma cells and catalyzes the formation of 3,3',4',5,7-pentahydroxyflavone (quercetin) into reactive quinone species and subsequent glutathionyl adducts. Therefore, we examined the effect of quercetin metabolism on the glutathione (GSH) bioreduction pathway and cell viability in DB-1 melanoma cells that express varying levels of tyrosinase (Tyr+). In a cell-free system, GSH was significantly decreased by quercetin, which coincided with the formation of glutathionyl adducts. In Tyr+ clones, quercetin decreased bioreduction capacity and increased reactive oxygen species (ROS) to a greater degree compared to control cells. The antioxidant/electrophile response element-induced enzymes, glutathione-S-transferase (GST), and nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 were expressed at high levels in Tyr+ cells and contributed to pro-oxidant quercetin metabolism. The basal level of ROS and apoptosis was higher in Tyr+ cells and were selectively increased after exposure to quercetin. The increase in apoptosis following quercetin exposure was p53/Bax mediated and correlated with a decrease in GST-driven bioreduction capacity and an increase in ROS. In conclusion, quercetin can selectively sensitize Tyr+ expressing melanoma cells to apoptosis and may serve as an adjuvant to chemotherapy by enhancing cell death and interfering with GST-mediated drug resistance.
• Humphries, M., Limesand, K., Schneider, J., Nakayama, K., Anderson, S., & Reyland, M. (2006). Suppression of apoptosis in the protein kinase Cδ null mouse in vivo. Journal of Biological Chemistry, 281(14). doi:10.1074/jbc.M507851200
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  Protein kinase C (PKC) δ is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used the PKCδ -/- mouse to ask if loss of PKCδ protects salivary glands against γ-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that γ-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater than 60% in the parotid glands of PKCδ-/- mice. Primary parotid cells from PKCδ-/- mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored by re-introduction of PKCδ by adenoviral transduction. Etoposide and γ-irradiation-induced activation of p53 is similar in primary parotid cells and parotid glands from PKCδ+/+ and PKCδ-/- mice, indicating that PKCδ functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal kinase is reduced in primary parotid cells from PKCδ-/- cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCδ. Similarly, c-Jun amino-terminal kinase activation is suppressed in vivo in γ-irradiated parotid glands from PKCδ-/- mice. These studies indicate an essential role for PKCδ downstream of the p53 response and upstream of the c-Jun amino-terminal kinase activation in DNA damage-induced apoptosis in vivo and in vitro. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
• Limesand, K., Schwertfeger, K., & Anderson, S. (2006). MDM2 is required for suppression of apoptosis by activated Akt1 in salivary acinar cells. Molecular and Cellular Biology, 26(23). doi:10.1128/MCB.01846-05
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  Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine18, total p53 protein accumulation, and p21WAF1 or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/ MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo. Copyright © 2006, American Society for Microbiology. All Rights Reserved.
• Limesand, K. H., Barzen, K. A., Sanders, L. A., Sclafani, R. A., Raynolds, M. V., Reyland, M. E., Anderson, S. M., & Quissell, D. O. (2003). Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture. In Vitro Cellular and Developmental Biology - Animal, 39(Issue 3-4). doi:10.1007/s11626-003-0012-1
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  Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 μM etoposide or 1.5 μM BFA inducing maximal caspase activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5′-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspase-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8-18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two- to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.
• Limesand, K. H., Higgs, S., Pearson, L. D., & Beaty, B. J. (2003). Effect of Mosquito Salivary Gland Treatment on Vesicular Stomatitis New Jersey Virus Replication and Interferon α/β Expression In Vitro. Journal of Medical Entomology, 40(Issue 2). doi:10.1603/0022-2585-40.2.199
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  The sensitivity of vesicular stomatitis (VS) viruses to Interferon (IFN)-mediated anti-viral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN α/β expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN α/β response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN α/β neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN α2 expression was reduced in L929 cells treated with SGH. Modulation of IFN α/β by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.
• Limesand, K. H., Higgs, S., Pearson, L. D., & Beaty, B. J. (2000). Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva. Parasite Immunology, 22(Issue 9). doi:10.1046/j.1365-3024.2000.00326.x
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  Saliva of arthropod vectors can modulate vertebrate host immunological functions in many ways. To investigate if vesicular stomatitis New Jersey Virus (VSNJ) infection could be potentiated by arthropod saliva, mice in three different age groups (3 days, 3 weeks, or > 8 months) were exposed to VSNJ-infected mosquitoes or were needle injected with an equivalent dose of VSNJ (titre 1.5-3 logs). Previous studies have demonstrated that VS viruses do not replicate in mice older than 3 weeks of age. Infection was monitored by examining serum for the presence of VSNJ at 2 days post-infection (PI) or for neutralizing antibody on days 7 and 14 PI. All 3-day-old mice succumbed to viral infection by mosquito transmission or delivery by injection. Ninety-four percent of the 3-week-old mice bitten by infected mosquitoes developed antibody, whereas antibody was detected in only 13% of inoculated mice. Adult mice developed neutralizing antibody (73%) when fed upon by infected mosquitoes, but only 11% developed antibody when virus was injected. Day 2 serum samples from 3-week and adult age groups were negative by virus isolation. These data indicate that mosquito mediated delivery of VSNJ exacerbates virus infection in mice older than 3 weeks.

Proceedings Publications

• Limesand, K. H., Nguyen, V. T., Ram, S., & Rodriguez, J. J. (2020). Combined Detection and Segmentation of Cell Nuclei in Microscopy Images Using Deep Learning. In 2020 IEEE Southwest Symposium on Image Analysis and Interpretation (SSIAI), 2020-, 26-29.
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  We propose a 3D convolutional neural network to simultaneously segment and detect cell nuclei in confocal microscopy images. Mirroring the co-dependency of these tasks, our proposed model consists of two serial components: the first part computes a segmentation of cell bodies, while the second module identifies the centers of these cells. Our model is trained end-to-end from scratch on a mouse parotid salivary gland stem cell nuclei dataset comprising 107 3D images from three independent cell preparations, each containing several hundred individual cell nuclei in 3D. In our experiments, we conduct a thorough evaluation of both detection accuracy and segmentation quality, on two different datasets. The results show that the proposed method provides significantly improved detection and segmentation accuracy compared to existing algorithms. Finally, we use a previously described test-time drop-out strategy to obtain uncertainty estimates on our predictions and validate these estimates by demonstrating that they are strongly correlated with accuracy.

Presentations

• Limesand, K. H. (2021). Wound healing as a framework for regeneration. International Association for Dental Research.
• Limesand, K. H. (2021). Wound healing as a framework for regeneration. NIH Salivary Gland Interest Group Meeting. virtual.
• Limesand, K. H. (2017, February). Signaling Mechanisms involved in Radiation-induced Chronic Loss of Function. Gordon Research Conference on Salivary Gland and Exocrine Research.

Poster Presentations

• Meeks, L., De Oliveira Pessoa, D., Martinez, J. A., Limesand, K. H., & Padi, M. (2021). Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction.. Cold Springs Harbor Network Biology conference. virtual.
• Gilman, K., Camden, J., Weisman, G., & Limesand, K. H. (2019, February). P2X7 receptor deletion suppresses γ-radiation induced hyposalivation. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Meyer, R., & Limesand, K. H. (2019, February). AMP-activated Protein Kinase Activation Impacts Acinar Cell Proliferation and Salivary Flow Rates following Radiation Therapy. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Wong, W. Y., Allie, S., & Limesand, K. H. (2019, February). Radiation-induced compensatory proliferation in parotid salivary glands is PKCζ-dependent and regulated by JNK signaling. Gordon Research Conference on Salivary Gland and Exocrine Research. Galveston, TX.
• Chibly, A., Ghosh, S., & Limesand, K. H. (2017, February). aPKCz Inhibits Nuclear Yap in Salivary Progenitors During Gland Regeneration. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Nguyen, V. T., & Limesand, K. H. (2017, February). Radiation treatment limits salisphere potential of parotid progenitor cells. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Wong, W. Y., & Limesand, K. H. (2017, February). Regulation of E-cadherin/β-catenin Interactions in Irradiated Salivary Glands. Gordon Research Conference on Salivary Gland and Exocrine Research.
• Limesand, K. H., & Chibly, A. (2015, Feb 14-18). Loss of Salivary Gland Cell Polarity due to Radiation. Gordon Research Conference on Salivary Gland and Exocrine Biology.
• Limesand, K. H., Nguyen, T., Chibly, A., & Harris, Z. (2015, Feb 14-18). Potential Role of Keratin 5 and 14 in Salisphere-Forming Ability of Adult Murine Parotid Glands after Radiation. Gordon Research Conference on Salivary Gland and Exocrine Biology.

Reviews

• Harris, Z., Donovan, M. G., Branco, G. M., Limesand, K. H., & Burd, R. (2016. Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy(p. 48).
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  Replacing current refractory treatments for melanoma with new prevention and therapeutic approaches is crucial in order to successfully treat this aggressive cancer form. Melanoma develops from neural crest cells, which express tyrosinase - a key enzyme in the pigmentation pathway. The tyrosinase enzyme is highly active in melanoma cells and metabolizes polyphenolic compounds; tyrosinase expression thus makes feasible a target for polyphenol-based therapies. For example, quercetin (3,3',4',5,7-pentahydroxyflavone) is a highly ubiquitous and well-classified dietary polyphenol found in various fruits, vegetables, and other plant products including onions, broccoli, kale, oranges, blueberries, apples, and tea. Quercetin has demonstrated antiproliferative and proapoptotic activity in various cancer cell types. Quercetin is readily metabolized by tyrosinase into various compounds that promote anticancer activity; additionally, given that tyrosinase expression increases during tumorigenesis, and its activity is associated with pigmentation changes in both early- and late-stage melanocytic lesions, it suggests that quercetin can be used to target melanoma. In this review, we explore the potential of quercetin as an anti-melanoma agent utilizing and extrapolating on evidence from previous in vitro studies in various human malignant cell lines and propose a "four-focus area strategy" to develop quercetin as a targeted anti-melanoma compound for use as either a preventative or therapeutic agent. The four areas of focus include utilizing quercetin to (i) modulate cellular bioreduction potential and associated signaling cascades, (ii) affect transcription of relevant genes, (iii) regulate epigenetic processes, and (iv) develop effective combination therapies and delivery modalities/protocols. In general, quercetin could be used to exploit tyrosinase activity to prevent, and/or treat, melanoma with minimal additional side effects.