Senescence in the Lab: Induction and Biomarkers

Scientists have become very interested in learning how cells in our body become senescent and understanding the resulting consequences of these cells to our health. To facilitate those studies, scientists can induce senescence in model cells and assess senescence using a variety of methods, such as qPCR, immunofluorescence, and the SA-βgal assay.

Throughout our lives, the cells in our bodies get damaged and do their best to repair themselves. When damage from either external or internal sources gets too bad, a cell’s DNA might be altered in a way that can lead to programmed cell death – or apoptosis, cancer – or unchecked proliferation, or senescence – a state of permanent cell cycle arrest.

Over the years, many research groups have become very interested in cellular senescence, specifically how cells around the body become senescent and the consequences of this cellular state for other cells and tissues. Before we explore the different ways scientists actually study senescent cells, let’s quickly review what we already know about them.

First, we know that cellular senescence can be induced by direct damage to a cell’s DNA, telomeres – or the protective caps at the end of DNA molecules, mitochondria, as well as alterations to epigenetic factors, which affect how our chromosomes are packaged and genes are expressed. Second, the leading hypothesis for why senescent cells exist is that they serve as a preventative measure against cancer. By arresting – or stopping – the cell cycle instead of devolving into unchecked proliferation, a senescent cell can be very helpful in preventing cancer. Third, researchers have also discovered that senescent cells secrete a variety of different molecules and proteins that can help with tissue repair in the short-term but can ultimately contribute to chronic inflammation when too many senescent cells accumulate around the body. These compounds are referred to as the senescence-associated secretory phenotype – or the SASP. There are many researchers working to determine if there are other ways that cellular senescence can be induced, how these cells contribute to disease, and what we might be able to do to intervene. So, now let’s take a closer look at how scientists actually study senescent cells.

Let’s say we are part of a research team testing various drug compounds for their ability to target senescent cells. It’s very difficult to see what senescent cells are doing inside the body, so for any senescence experiment we have to rely on models that can be studied in the lab. The term “model” can refer to both model organisms, like mice or fruit flies, or it can mean cellular models, where specific types of cells are grown in the lab to be used in various experiments (fibroblasts).

Let’s say our team is conducting a preliminary screening for new drug candidates, so we begin by testing a panel of drugs on a cell model before moving to a model organism. The results of these experiments can help us understand how the same types of cells that exist in the body might behave or respond to different conditions. In any experiment involving senescent cells, we have to start by creating them in a laboratory setting. It isn’t realistic to isolate lots of senescent cells from human tissue, so we have to start with a good model that closely approximates actual senescent cells. Many researchers use fibroblasts as a cell model in different experiments. These cells are found all across the body and their main job is to produce collagen which helps form the structure of our organs and tissues. These cells are commonly used in experiments with senescent cells because they are easy to grow and manipulate in a laboratory setting, and they are found all over the body.

To begin our experiment, we obtain our fibroblasts and, after allowing the cells to grow for a few days, we then start the process of senescence induction – or turning the fibroblasts into actual senescent cells. So how are senescent cells created in a lab? Well, we know that senescence cells accumulate in the body because of DNA damage, so when senescent cells are created in the lab, researchers often try to recreate this process by inducing DNA damage or altering gene expression in the cells. This can be done using chemical compounds, radiation, or sometimes scientists will simply allow the population of cells to grow until they reach a point called replicative senescence – or the limit where they will not divide anymore.

For our experiment, we choose to use a chemical called doxorubicin, which is commonly used in the field to induce senescence in our fibroblasts. It’s important to split the population of fibroblasts and induce senescence in one group, while another group serves as the control to make sure any differences in the two populations are actually due to the treatment and not other random variables. After exposing one group to doxorubicin, we then let both groups of cells grow for around seven to ten days. Then, after patiently waiting for over a week, we can check and make sure that the cells have actually become senescent. It is essential to confirm that the model of cellular senescence has worked effectively. If the model hasn’t worked properly, the rest of the experiment won’t be reliable.

So, how do we know if the cell model has worked? Well, let’s think back to the major hallmarks of senescence: significant DNA damage, a permanent arrest of the cell cycle, and the senescence-associated secretory phenotype – or the SASP. If we see evidence of each of these hallmarks in our senescent cell population, but not in our control population, we can be confident that we have successfully generated a good cell model which can be used in our preliminary drug screen.

To assess the first hallmark of senescence, DNA damage, we can use staining techniques or quantitative real time PCR – also called qPCR – to look for evidence that the cells have tried to repair their DNA after exposure to doxorubicin. Cells rely on specific proteins to repair breaks or mutations in DNA, so we can look for proteins like γ-H2AX, HMGB1, and other proteins involved in DNA repair. It can also be helpful to look for changes in the localization of these proteins. For example, HMGB1 is typically found in the nucleus of healthy cells, but in senescent cells this protein can be seen in the cytoplasm as well as the nucleus.

In our experiment, we use staining techniques to look at the localization of HMGB1. We use a blue stain to mark the nucleus of individual cells and we use a red stain to mark the HMGB1 proteins in the cell. In our non-senescent cells, we can see that the red and blue stains have mixed to create a purple color. This indicates that HMGB1 is in the nucleus, which is what we expect in healthy cells. In our doxorubicin-treated cells, we see that the red stain is showing up in the cytoplasm of the cell indicating that some of the HMGB1 proteins are leaving the nucleus. This result indicates that our treated cells have been working to repair their DNA, but the cell is getting overly stressed by the amount of damage, so the repair proteins are leaving the nucleus, which supports the conclusion that the doxorubicin treatment was effective in creating senescent cells.

Next, we’ll assess the second hallmark of senescence – a permanent arrest of the cell cycle. The cell cycle is controlled by a complex series of protein interactions that happen in the cytoplasm of the cell. These interactions are known as a signaling network or pathway. When a cell becomes senescent, changes occur in this signaling network and we can use qPCR to see how the expression of some of the most important cell cycle genes have changed. Genes like p16, p21, and p53 code for proteins that are important cell cycle regulators and scientists have discovered that their expression increases in senescent cells.

In our experiment, we take a small number of cells from the control group and the senescent group and use them in a qPCR assay to compare the expression of cell cycle genes. If we analyze our qPCR data and plot it in a bar graph, we can see that the expression of these cell cycle genes is significantly increased in the senescent cell population. Again, this supports the conclusion that our senescent cell model has worked effectively.

Next, we’ll assess the third hallmark of senescence – the SASP. Remember that the SASP is made up of different proteins and molecules that both aid in tissue repair but can also contribute to chronic inflammation. When confirming whether senescence induction has worked, we can check for the expression of some core SASP factors. For our experiment, we will assess whether the senescent cells express inflammatory factors, like interleukin 6, also known as IL-6, as well as a type of molecule called a chemokine, which specifically helps recruit immune cells to the site of cell damage or infection. Interleukin 8, or IL-8, is an example of a molecule in the chemokine family. There are different types of chemokines, but in general, these molecules help recruit immune cells to the site of inflammation and are core proteins that make up the SASP. We run a qPCR experiment to look for SASP markers and we see that the senescent cells show significantly higher expression of IL-6 and IL-8. This result provides yet another piece of evidence that our senescence model has worked effectively.

Lastly, as scientists have conducted more experiments on senescent cells, they found that these cells tend to have more of a protein called beta-galactosidase relative to non-senescent cells. This protein is found in the lysosome and helps break down carbohydrates containing the sugar galactose. The enzyme cleaves the bond between the sugar molecules to break down the larger macromolecule. While this protein is found in non-senescent cells, researchers have discovered that there is a significant accumulation of this protein in senescent cells. This has allowed for the creation of a widely used technique to identify senescent cells. Researchers often use a technique called the senescence-associated beta-galactosidase assay – or SA-βgal assay – to assess senescence induction. In our fibroblast experiment, we expose our senescent and non-senescent cells to a molecule called X-gal. The beta-galactosidase enzymes bind to X-gal and cleave it. Instead of breaking down a large carbohydrate molecule, this cleavage results in the formation of a blue dye which can be visualized under a microscope. When we compare the presence of the blue dye in our senescent and non-senescent cell populations, we observe a significantly greater number of blue cells in the senescent group than in the control. It is normal for there to be a few blue cells in the control group, but the difference should still be quite clear by eye. We can quantify the percentage of senescent cells in each population to get an estimate of how well our senescence induction worked.

Together, all of these different methods of measuring senescence induction help our research team gain a more complete picture of the cell model we’ve created. With any senescence experiment, it is important to use multiple analysis methods to assess senescence induction. We do not yet have one perfect marker or assay to identify senescent cells, so we have to rely on multiple methods, like qPCR, SA-βgal, and staining techniques to confirm our senescence model. Once this important step of verifying the model has been completed, we can move on to testing potential drug compounds for their ability to target senescent cells or we could pursue other research avenues, such as investigating novel SASP factors or designing a cell therapy to target senescent cells. These experiments could help advance our understanding of how these cells contribute to various diseases and what we might be able to do to intervene.

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