Methods of Induction of Cellular Senescence
Poster #: 125
Session/Time: B
Author:
Sara Brooke Palega, BS, MS
Mentor:
Yan Y Sanders, MD
Research Type: Basic Science
Abstract
INTRODUCTION:
Idiopathic Pulmonary Fibrosis (IPF) is a fatal, age-related interstitial lung disease with no curative treatment. IPF arises from aberrant wound healing that leads to excessive extracellular matrix deposition, which makes breathing increasingly difficult as the disease progresses. The accumulation of senescent cells with abnormal secretory phenotypes exacerbates inflammation and impairs tissue repair. This altered state, known as the senescence-associated secretory phenotype (SASP), drives fibrosis by promoting epithelial dysfunction, fibroblast activation, and pathological ECM remodeling. Therefore, eliminating senescent cells has emerged as a potential therapeutic option. However, cellular senescence caused by various stimuli exhibit different secretory phenotypes; this heterogeneity makes it challenging to study lung fibrosis, as it is not clear whether cells would present the same mechanisms under senescence induced by different stimuli. In this project, we used various methods to induce senescence in IMR90 human lung fibroblast cells, including replicative, genotoxic stress, such as X-ray and UV radiation. The senescent markers of p16 and p21, as well as SASP related chronic inflammatory markers such as IL-1β, IL-6, and IL-8, will be evaluated at RNA and protein levels of expression. Additionally, after establishing the senescent models, senolytics will be tested for their ability to clear senescent fibroblast cells.
METHODS:
Methods of inducing cellular senescence were tested by exposing the human lung IMR90 cell line to acute X-ray radiation once at 2Gy or 8Gy, repeated at 1Gy every 2-3 days for a total of 10 days, or subjecting to UV treatment once at 10mJ/cm^2 or 15mJ/cm^2. The cells were collected after 15 days of final treatment for RNA and protein extractions, as well as senescence-associated β-gal staining to examine cell phenotype. Quantitative real-time PCR (qPCR) was used to evaluate the expression of the senescent and SASP markers including p21, p16, IL-1β, IL-6, and IL-8; the protein levels expression was assessed with western blots or ELISA assays.
RESULTS:
Acute and repeated low dose X-ray radiation showed increased β-gal stain, and increase the expression of senescence and SASP markers, like p16, p21, IL-1β, IL-6, and IL-8 in IMR90 human lung fibroblasts. UV treated cells showed increased p21. This project is ongoing, and we are not sure if all, or only some markers, will be altered. It is expected that these effects will be lessened by treatment with senolytics, which eliminate senescent cells.
CONCLUSION:
The various methods to induce senescence in IMR90 cells were evaluated by senescence-associated β-gal staining, as well as well-established senescent markers p21, p16, and SASP-related genes IL-1β, IL6 and IL8. After establishing these models, the mechanisms of induced senescence will be explored, and the effectiveness of senolytics will be assessed. Overall, this project will lead to a better understanding of how environmental stress leads to cellular senescence, provide better in vitro aging lung injury repair models that could lead to stronger medical interventions for patients with IPF.
Idiopathic Pulmonary Fibrosis (IPF) is a fatal, age-related interstitial lung disease with no curative treatment. IPF arises from aberrant wound healing that leads to excessive extracellular matrix deposition, which makes breathing increasingly difficult as the disease progresses. The accumulation of senescent cells with abnormal secretory phenotypes exacerbates inflammation and impairs tissue repair. This altered state, known as the senescence-associated secretory phenotype (SASP), drives fibrosis by promoting epithelial dysfunction, fibroblast activation, and pathological ECM remodeling. Therefore, eliminating senescent cells has emerged as a potential therapeutic option. However, cellular senescence caused by various stimuli exhibit different secretory phenotypes; this heterogeneity makes it challenging to study lung fibrosis, as it is not clear whether cells would present the same mechanisms under senescence induced by different stimuli. In this project, we used various methods to induce senescence in IMR90 human lung fibroblast cells, including replicative, genotoxic stress, such as X-ray and UV radiation. The senescent markers of p16 and p21, as well as SASP related chronic inflammatory markers such as IL-1β, IL-6, and IL-8, will be evaluated at RNA and protein levels of expression. Additionally, after establishing the senescent models, senolytics will be tested for their ability to clear senescent fibroblast cells.
METHODS:
Methods of inducing cellular senescence were tested by exposing the human lung IMR90 cell line to acute X-ray radiation once at 2Gy or 8Gy, repeated at 1Gy every 2-3 days for a total of 10 days, or subjecting to UV treatment once at 10mJ/cm^2 or 15mJ/cm^2. The cells were collected after 15 days of final treatment for RNA and protein extractions, as well as senescence-associated β-gal staining to examine cell phenotype. Quantitative real-time PCR (qPCR) was used to evaluate the expression of the senescent and SASP markers including p21, p16, IL-1β, IL-6, and IL-8; the protein levels expression was assessed with western blots or ELISA assays.
RESULTS:
Acute and repeated low dose X-ray radiation showed increased β-gal stain, and increase the expression of senescence and SASP markers, like p16, p21, IL-1β, IL-6, and IL-8 in IMR90 human lung fibroblasts. UV treated cells showed increased p21. This project is ongoing, and we are not sure if all, or only some markers, will be altered. It is expected that these effects will be lessened by treatment with senolytics, which eliminate senescent cells.
CONCLUSION:
The various methods to induce senescence in IMR90 cells were evaluated by senescence-associated β-gal staining, as well as well-established senescent markers p21, p16, and SASP-related genes IL-1β, IL6 and IL8. After establishing these models, the mechanisms of induced senescence will be explored, and the effectiveness of senolytics will be assessed. Overall, this project will lead to a better understanding of how environmental stress leads to cellular senescence, provide better in vitro aging lung injury repair models that could lead to stronger medical interventions for patients with IPF.