Browsing by Author "Zhou, Huaxin"

Now showing 1 - 7 of 7
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    DNA Repair Capacity for Personalizing Risk and Treatment Response - Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs)
    (Elsevier, 2022) Al Nasrallah, Nawar; Zhou, Huaxin; Smith, Patricia A.; Sears, Catherine R.; Medicine, School of Medicine
    DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.
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    JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling
    (Wiley, 2020-07) Lü, Lanhai; Yakoumatos, Lan; Ren, Junling; Duan, Xiaoxian; Zhou, Huaxin; Gu, Zhen; Mohammed, Muddasir; Uriarte, Silvia M.; Liang, Shuang; Scott, David A.; Lamont, Richard J.; Wang, Huizhi; Medicine, School of Medicine
    Homeostasis between pro- and anti- inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, proinflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen P. gingivalis enhances the activity of JAK3 in innate immune cells, and subsequently phospho-inactivates Nedd4-2, a ubiquitin E3 ligase. In turn, Wnt3 ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in proinflammatory cytokine levels. In contrast, JAK3 inhibition or Wnt3a robustly enhances NF-κB activity and the production of proinflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gain- and loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and β-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P. gingivalis-infected gingival tissues, and increases disease severity. Together, our results reveal a new anti-inflammatory role for JAK3 in innate immune cells and show that the underlying signaling pathway involves Nedd4-2-mediated Wnt3a ubiquitination.
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    Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development
    (MDPI, 2021-11-06) Mickler, Elizabeth A.; Zhou, Huaxin; Phang, Tzu L.; Geraci, Mark W.; Stearman, Robert S.; Sears, Catherine R.; Medicine, School of Medicine
    Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer.
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    Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9
    (Springer Nature, 2022-07-01) Fu, Yongyao; Pajulas, Abigail; Wang, Jocelyn; Zhou, Baohua; Cannon, Anthony; Cheung, Cherry Cheuk Lam; Zhang, Jilu; Zhou, Huaxin; Fisher, Amanda Jo; Omstead, David T.; Khan, Sabrina; Han, Lei; Renauld, Jean-Christophe; Paczesny, Sophie; Gao, Hongyu; Liu, Yunlong; Yang, Lei; Tighe, Robert M.; Licona-Limón, Paula; Flavell, Richard A.; Takatsuka, Shogo; Kitamura, Daisuke; Sun, Jie; Bilgicer, Basar; Sears, Catherine R.; Yang, Kai; Kaplan, Mark H.; Microbiology and Immunology, School of Medicine
    Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c- interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r-/- mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.
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    Xeroderma Pigmentosum Group C Deficiency Alters Cigarette Smoke DNA Damage Cell Fate and Accelerates Emphysema Development
    (American Thoracic Society, 2018-03) Sears, Catherine R.; Zhou, Huaxin; Justice, Matthew J.; Fisher, Amanda J.; Saliba, Jacob; Lamb, Isaac; Wicker, Jessica; Schweitzer, Kelly S.; Petrache, Irina; Medicine, School of Medicine
    Cigarette smoke (CS) exposure is a major risk factor for the development of emphysema, a common disease characterized by loss of cells comprising the lung parenchyma. The mechanisms of cell injury leading to emphysema are not completely understood but are thought to involve persistent cytotoxic or mutagenic DNA damage induced by CS. Using complementary cell culture and mouse models of CS exposure, we investigated the role of the DNA repair protein, xeroderma pigmentosum group C (XPC), on CS-induced DNA damage repair and emphysema. Expression of XPC was decreased in mouse lungs after chronic CS exposure and XPC knockdown in cultured human lung epithelial cells decreased their survival after CS exposure due to activation of the intrinsic apoptosis pathway. Similarly, cell autophagy and apoptosis were increased in XPC-deficient mouse lungs and were further increased by CS exposure. XPC deficiency was associated with structural and functional changes characteristic of emphysema, which were worsened by age, similar to levels observed with chronic CS exposure. Taken together, these findings suggest that repair of DNA damage by XPC plays an important and previously unrecognized role in the maintenance of alveolar structures. These findings support that loss of XPC, possibly due to chronic CS exposure, promotes emphysema development and further supports a link between DNA damage, impaired DNA repair, and development of emphysema.
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    XPC Protects against Carcinogen-Induced Histologic Progression to Lung Squamous Cell Carcinoma by Reduced Basal Epithelial Cell Proliferation
    (MDPI, 2024-04-13) Sears, Catherine R.; Zhou, Huaxin; Hulsey, Emily; Aidoo, Bea A.; Sandusky, George E.; Al Nasrallah, Nawar; Medicine, School of Medicine
    Lung squamous cell carcinoma (LUSC) is the second leading cause of lung cancer. Although characterized by high DNA mutational burdens and genomic complexity, the role of DNA repair in LUSC development is poorly understood. We sought to better understand the role of the DNA repair protein Xeroderma Pigmentosum Group C (XPC) in LUSC development. XPC knock-out (KO), heterozygous, and wild-type (WT) mice were exposed topically to N-nitroso-tris-chloroethylurea (NTCU), and lungs were evaluated for histology and pre-malignant progression in a blinded fashion at various time-points from 8-24 weeks. High-grade dysplasia and LUSC were increased in XPC KO compared with XPC WT NTCU mice (56% vs. 34%), associated with a higher mean LUSC lung involvement (p < 0.05). N-acetylcysteine pre-treatment decreased bronchoalveolar inflammation but did not prevent LUSC development. Proliferation, measured as %Ki67+ cells, increased with NTCU treatment, in high-grade dysplasia and LUSC, and in XPC deficiency (p < 0.01, ANOVA). Finally, pre-LUSC dysplasia developed earlier and progressed to higher histologic classification sooner in XPC KO compared with WT mice. Overall, this supports the protective role of XPC in squamous dysplasia progression to LUSC. Mouse models of early LUSC development are limited; this may provide a valuable model to study mechanisms of LUSC development and progression.
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    XPC protects against smoking- and carcinogen-induced lung adenocarcinoma
    (Oxford University Press, 2019-04-10) Zhou, Huaxin; Salib, Jacob; Sandusky, George E.; Sears, Catherine R.; Medicine, School of Medicine
    Cigarette smoke (CS) contains hundreds of carcinogens and is a potent inducer of oxidative and bulky DNA damage, which when insufficiently repaired leads to activation of DNA damage response and possibly mutations. The DNA repair protein xeroderma pigmentosum group C (XPC) is primed to play an important role in CS-induced DNA damage because of its function in initiating repair of both bulky oxidative DNA damage. We hypothesized that loss of XPC function will increase susceptibility to developing CS- and carcinogen-induced lung cancer through impaired repair of oxidative DNA damage. Mice deficient in XPC (XPC-/-) exposed to chronic CS developed lung tumors whereas their wild-type littermates (XPC+/+) did not. XPC-/- mice treated with the CS-carcinogen urethane developed lung adenocarcinomas representing progressive stages of tumor development, with lung tumor number increased 17-fold compared with XPC+/+ mice. Mice heterozygous for XPC (XPC+/-) demonstrated a gene-dose effect, developing an intermediate number of lung tumors with urethane treatment. Treatment of XPC-/- mice with the carcinogen 3-methylcholanthrene followed by the proliferative agent butylated hydroxytoluene resulted in a 2-fold increase in lung adenocarcinoma development. Finally, tumor number decreased 7-fold in the lungs of XPC-/- mice by concurrent treatment with the antioxidant, N-acetylcysteine. Altogether, this supports a mechanism by which decreased XPC expression promotes lung adenocarcinoma development in response to CS-carcinogen exposure, due in part to impaired oxidative DNA damage repair.