Assay Development to Monitor Cell Senescence

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Senescence is a process of aging within cells. We developed a number of bioassays to evaluate anti-aging activity of new compounds. Initially in high throughput and subsequently in more hits-to-lead mechanistic assays.

Challenges

We wanted to examine the effect of the test molecules in cellular models of senescence. To achieve this, we developed several assays and used positive control compounds to demonstrate the validity of these assays.

What We Did...

Cellular senescence is defined as irreversible cell cycle arrest that can occur in response to intra- or extra-cellular perturbations and is often linked to aging. Senescence is highly regulated and can be beneficial (foetal development and tumour suppression) or detrimental (premature-aging diseases and tissue remodelling). 

There are several techniques used to study senescence including investigating expression of senescence-associated β-galactosidase and other senescent specific markers, examining cell cycle progression that leads to a reduction in cell proliferation and downstream orthogonal readouts.

Senescence assay development

Figure. 1. Senescence can be induced in a several ways, including via DNA damage, oxidative stress, or replicative exhaustion.1

Examining Cell Cycle Progression Through Protein Phosphorylation

Cell cycle progression is regulated by a family of protein kinase. Simplistically, in normal proliferating cells, cyclin-dependent kinase 4 and 6 (CDK4/6) bind to and form complexes with cyclin-D. This complex phosphorylates retinoblastoma (Rb) protein to allow Rb to dissociate from the transcription factor E2F. 

E2F is then able to translocate to the nucleus, upregulating expression of cell cycle progression genes from G1 to S-phase. p21 is a potent cyclin-dependent kinase inhibitor. p21 binds to and inhibits the activity of CDK4/6, thus preventing Rb phosphorylation and halting cell cycle progression. 

The elevation of p21 and reduction of phospho-Rb can be indicative of senescence as demonstrated in figure 2 using Doxorubicin and Bleomycin in WI-38 cells.

Senescence assay development
Senescence assay development
Senescence assay development
Senescence assay development

Figure. 2. Protein expression and phosphorylation: Cell cycle progression proteins were probed using the Bio-Techne JESS system, a high-throughput immunoblotting instrument, in lysates from WI-38 cells (left) and A549 cells (right). Cells were treated with Doxorubicin or Bleomycin for 24 hours prior to probing for p21 and Phospho-Rb.

Senescence Associated Secretory Phenotype (SASP) Observed Though Kinectic Imaging of Cell Infill

Senescent cells are metabolically active and can demonstrate significant changes in their secretome by secreting a plethora of soluble signalling factors that can cause a chain reaction and induce senescence in neighboring cells. To study this effect, we developed a Scratch Wound Assay using the Incucyte SX5 kinetic imager in fibroblast cells +/- compounds.

Senescence assay development
Senescence assay development
Senescence assay development

Figure. 3. Scratch Wound Assay: Identical scratches were made in monolayer cultures of untreated fibroblasts. In other wells, fibroblasts were subject to conditioned media collected from cells that had been induced into senescence with Doxorubicin, Doxorubicin plus Quercetin, Cytochalasin D or DMSO. Media on scratched cells was changed with conditioned media and closure of the scratch was tracked over 72-hours using the Incucyte SX5 kinetic imager.

Wound closure occurred rapidly in DMSO conditioned wells, whereas both Doxorubicin and Cytochalasin D conditioned media decreased wound closure and Quercetin was able to antagonize the effect of Doxorubicin (see images of cells at 24- and 72- hours).

Examining Senescence and Cell Health

Senescent cells possess β-galactosidase activity termed “senescence-associated β-galactosidase” (SA-β-Gal). This is present in senescent but not pre-senescent or quiescent cells. SA-β-Gal catalyzes the hydrolysis of β-galactosides into monosaccharides and can be detected by measuring the fluorescent cleavage product (4-MU), formed when SA-β-Gal catalyzes a chemical 4-MUG (A) and detectable at an excitation wavelength of 360nm.

Senescence assay development
Senescence assay development

Figure. 4. Human fibroblasts were treated with Doxorubicin, leading to an increase in SA-b-Gal activity in a dose-dependent manner concomitant with cellular senescence.

Senescence assay development

Figure. 5. Bleomycin was used as a positive control to determine the extent of senescence induction by other compounds in different cellular backgrounds; WI-38 and A549.

Senescent cells undergo permanent cell cycle arrest at the G1 phase, subsequently causing a reduction in the overall proliferation of a cell population but without triggering apoptosis. 

Senescence assay development
Senescence assay development

Figure. 6. Fibroblasts treated with DMSO or Doxorubicin and imaged for 7-days using the Incucyte SX5 live cell imager.

Figure. 7. RealTime-Glo™ Assay in fibroblasts treated with DMSO or Doxorubicin. RealTime-Glo™ detects ATP generation and turnover as a biomarker of the number of viable cells in a population, by measuring the reducing potential of cells and thus metabolism. Proliferating cells show an increase in luminescence over time.

Summary

We successfully established a multi-pronged approach for investigating senescence using several technologies. Some of these can be performed in a high-throughput manner, while others offering a more technical and mechanistic insights. This approach allowed the test molecules to be screened in an orthogonal manner, screening a large number of compounds in higher throughput and prosecuting the output of the screen in a more mechanistic manner for senolytic activity, to accelerate anti-aging drug discovery and pave the way for future research in the field of chemotherapeutics.

References

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