Investigating Cellular Senescence Following Radiation Exposure

Stephen Liu, Mihai Dumbrava, Adrienne Wan and Laura Paterson UNRESTRICTED / ILLIMITÉ

Cellular Senescence

Cellular senescence refers to a process in which cell growth and development are irreversibly halted.

No known factor can induce senescent cells to re-enter the cell cycle.

Although they no longer divide, senescent cells remain metabolically active.

UNRESTRICTED / ILLIMITÉ

Types of Senescence

Replicative Senescence Stress Induced

Premature Senescence (SIPS)

UNRESTRICTED / ILLIMITÉ

Gamma Radiation

Gamma (γ) radiation is a form of indirectly ionizing radiation.

It can damage DNA through two different processes:1. Direct Action2. Indirect Action

UNRESTRICTED / ILLIMITÉ

Lymphocytes

Lymphocytes are a type of white blood cell which recognize and respond to particular pathogens. They are one of the most radiosensitive and long-lived cell types in the human body.

UNRESTRICTED / ILLIMITÉ

Gamma Radiation Doses

The Lethal Dose 50 (LD50) is between 4 to 7 Gy for humans (1 Gy= 1 J/kg).

UNRESTRICTED / ILLIMITÉ

Absorbed Gamma Radiation Dose (Gy)

Irradation Time (s) Dose Rate (Gy/s) Blood Volume (mL)

0 - - 2.5

0.5 3.4 0.0607 2.5

0.75 7.5 0.0607 2.5

1 11.6 0.0607 2.5

2 28 0.0611 2.5

3 44.4 0.0611 2.5

4 60.8 0.0611 2.5

SIPS Pathway

UNRESTRICTED / ILLIMITÉ

Metabolic processes

Gamma radiation exposure

DNA damage which can lead to senescence

Biomarkers of Senescent Cells

UNRESTRICTED / ILLIMITÉ

Purpose

This project sets out to determine a relationship between the amount of absorbed gamma radiation and the occurrence of cellular senescence.

Absorbed Gamma Radiation

Perc

enta

ge o

f Se

nesc

ent c

ells

UNRESTRICTED / ILLIMITÉ

Hypothesized Correlation

Procedure – Isolating Lymphocytes

UNRESTRICTED / ILLIMITÉ

• Irradiate human blood samples

• Dilute blood using PBS

• Carefully layer diluted blood samples on Ficoll

• Centrifuge to separate blood into different layers

• Wash lymphocytes, transfer into RPMI Media

• Incubate for 48 hours at 37°C, 5% CO2

Procedure – Introducing p16 Antibody

UNRESTRICTED / ILLIMITÉ

Ethanol Permeabilization

• Vortex and add ethanol to cell pellet

• Incubate at -20°C for at least 2 hours

• Add p16 antibody into cell suspension

• Incubate for 20-30 minutes in the dark

Flow Cytometry

1. Cell sample is inserted into flow cytometer through a small nozzle

2. Cells move in a single file past lasers

3. p16 probes are excited by these lasers to emit light in a specific channel

4. Detectors measure the light emitted

5. The amount of cells, as well as their morphological and photometric features, is determined

UNRESTRICTED / ILLIMITÉ

p16 probes

UNRESTRICTED / ILLIMITÉ

Phycoerythrin (PE) fluorochrome – a chemical that fluoresces to distinguish cellular properties.

p16 antibody

Normal Cell – Photometrically non-senescent

Fluorescent Cell – Photometrically and morphologically senescent

IDEAS® Software Analysis

Cell features are plotted on different graphs to distinguish senescence.

The percentage of senescent cells can be calculated using the equation:

UNRESTRICTED / ILLIMITÉ

Experimental Results

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50

0.5

1

1.5

2

2.5

3

3.5

4

f(x) = 0.142849977797818 x² − 0.0740297450563477 x

Effects of Radiation on Cellular Senescence γ

Radiation Dose (Gy)γ

Per

cen

tage

of S

enes

cen

t Ce

lls (

%)

Discussion – Acute Radiation SyndromeSe

nesc

ence

in %

Error Analysis

Laboratory Procedure

Software Analysis

Blood Donors

Limited Sample

Size

• Sample contamination

• Cell loss• Incorrect

morphology

• Different radiation exposure

• Lifestyle choices

• Insufficient data to draw conclusions

UNRESTRICTED / ILLIMITÉ

Application

THE BRIGHT SIDE

Tumor Suppression

Cellular senescence can stop the uncontrolled division of cancerous

cells

Aging

Senescent cells can disrupt cell growth, differentiation, and initiate or promote age-related diseases

UNRESTRICTED / ILLIMITÉ

THE DARK SIDE

Future Work

Radiation Therapy – determining the amount of radiation which will maximize the occurrence of cellular senescence.

Cell Repair

Cell Misrepair

Cell Death

Normal Cell

Division

Determining Accidental Radiation Dose

Conclusion

There is a positive quadratic correlation between the absorbed gamma radiation dose and the percentage of senescent lymphocytes.

Ranging from radiation therapy to age-related diseases, cellular senescence has a variety of applications and drawbacks.

This research has created a foundation for diagnosing an individual’s original radiation dose through the analysis of senescent cells.

Acknowledgements

Works Cited

Abcam. "Introduction to Flow Cytometry." Introduction to Flow Cytometry. Abcam, 2014. Web. 30 July 2014.

AbD Serotec. "Fluorochromes and Light." Fluorochromes and Light. Bio-Rad Laboratories, 2014. Web. 01 Aug. 2014.

Hall, Eric J., and Amato J. Giaccia. Radiobiology for the Radiologist. Philadelphia: Lippincott Williams & Wilkins, 2006. Print.

International Atomic Energy Agency. Radiation Biology: A Handbook for Teachers and Students. Vienna: International Atomic Energy Agency, 2010. IAEA. International Atomic Energy Agency, 2010. Web. 1 Aug. 2014.

Jefferson Lab. "Radiation Biological Effects." Radiation Biological Effects. Jefferson Lab, n.d. Web. 10 July 2014.

Rahman, Misha. "Introduction to Flow Cytometry." Introduction to Flow Cytometry. Bio-Rad Laboratories, 2014. Web. 24 July 2014.

Rodier, Francis, and Judith Campisi. "Four Faces of Cellular Senescence." Four Faces of Cellular Senescence. The Rockefeller University Press, 14 Feb. 2011. Web. 24 July 2014.

Tough, D. F., and J. Sprent. "Lymphocyte life-span and memory." National Center for Biotechnology Information. U.S. National Library of Medicine, May 1995. Web. 01 Aug. 2014.

The End

Thank you for listening!Are there any questions?