A short presentation for Jessica Bevins's High School class on application of cell biology concepts they are learning

2. What education/training after high school does it take to
become a (life) scientist?
General Path What I Did
• Bachelors degree (~4yrs) in a science • Bachelors degree (in Finance) but
as part of a individually developed
curriculum took enough biology and 2000-2004
• (Only sometimes) Masters degree 1- chemistry to get 2 minors
2yrs, classes & perform focused
research project
• PhD degree, some classes and full-time • PhD in Molecular Carcinogenesis
research (have to discover something at the UTHSC/MDACC, Smithville 2004-2011
NEW!), usually paid for RA/TA, 5-7+yrs, TX
write a 100-200 page dissertation at end
• Postdoctoral fellowship(s) 2-5+ yrs – full • TRIUMPH postdoctoral fellow, at
time research, learning how to write the University of Texas MD 2011-now
grants, more research training, how to Anderson Cancer Center, Houston
run a lab etc TX

3. Education in pictures
Newark DE
Smithville (1hr from
Austin), TX Houston, TX

4. Cancer – the disease which affects nearly 2M
Americans, and causes >500K deaths/year
• What is cancer?
• Cells that acquire mutations in their DNA that
result in abnormal growth – divide continually
when they are not supposed to, and don’t die
when they receive death signals.
• Tumors form when cancer cells divide and
form a mass.
• Tumors grow their own blood vessels to get
nutrients to survive (angiogenesis)
• Soon cancer cells become invasive – take
over the normal tissue, and spread around the
body making metastases (e.g. in the lungs, or
brain or bones). This is why people die from
cancer – usually primary tumors do not kill
people.
Cancer biologists like to compare
cancer cells and normal cells to
understand what went wrong, and how
to kill the cancer cells alone!

5. Tools of the trade – examples of different breast cancer cells taken
from human patients growing on plastic plates in lab
• Cells can be
cultured from
humans, mice,
other rodents,
insects etc….
• We can culture
cancer cells
indefinitely in
solutions of
glucose and
amino acids (to
make proteins)!

6. Proteins function in pathways in cells
Challenge is there are
100s of these
pathways & they are
all interconnected in
ways we don’t fully
understand!
Some proteins have
different functions
depending on
WHERE they are and
what proteins they
interact with!

7. PhD Thesis: “ATM signaling to TSC2: Mechanisms and
Implications for Cancer Therapy”
Key questions:
• How do cells (including cancer cells) detect and respond to DNA and
oxidative damage?
• Are these protein pathways linked to cell survival or death?
• Can we target these survival pathways to improve cancer therapy
(chemotherapy, radiation, big surgeries)?
Methods overview:
• Study protein localization in the cell and their function (for example whether they are active
or not) using fluorescence microscopy and cell fractionation
• Used genetic approaches to study whether a process depends on a single protein – used
cells that either lack the protein, or used a technique called RNA interference to reduce
the amount of the protein I was interested in.
• Used drugs to inhibit pathways – such as protein export from the nucleus

8. Examples of techniques used in a cell biology lab
Western blotting – we make protein mixtures from cells or animal tissues, and run
gels to separate the proteins based on size. Using antibodies to proteins we can
measure amounts of these proteins and their modifications.
We can also break up cells and use selective solutions of chemicals (buffers) to only
get cytoplasm, membranes or nucleus

9. Supplementary Figure S3
Techniques in a cell biology lab (cont.)
a
HEK 293 cells
b
HEK 293
IgG WT RQ RW RG IgG WT RQ
PEX5 (light) Flag-TSC2
PEX5 (dark) IP: Flag
Fluorescence and confocal microscopy – use this to observe
Flag-TSC2
PEX5
protein localization (what organelles are important?)
c e
Flag PMP70 Merge/DAPI
HEK
WT
IgG WT RQ
Flag-TSC2
Myc-TSC1
Flag PMP70 Merge/DAPI
Myc-TSC1
RQ
Flag-TSC2
HeLa cells
Flag PMP70 Merge/DAPI
RG
Flag PMP70 Merge/DAPI
RW
d
Flag Calnexin Merge/DAPI
RQ
ells

10. More cell biology techniques
Electron microscopy – observe structural Animal/human tissue studies
information inside cells without using – immunohistochemistry
antibodies to detect individual proteins staining for proteins using
antibodies then chemical
reactions that generate
brown color

11. Major findings of my PhD work (team project)
• Key discovery – a very important protein (called ATM) plays different
functions depending on where it is localized in the cell
• Before my work, most people thought it mainly functioned to
sense DNA damage in the nucleus, and recruit other proteins to
REPAIR the damage or trigger cell death if too much damage.
• I showed convincingly that a separate pool of this protein that
didn’t have to leave the nucleus could respond to a oxidative
damage and trigger autophagy
• Autophagy is a recycling process cells use to degrade damaged
organelles and certain proteins
• Often used by cancer cells to survive stresses such as
chemotherapy and radiation.
• Understanding how and when this applies can help us design
ways to block cancer cell survival.

13. Cancer and the cell cycle
• Cell cycle deregulation is virtually
universal feature in cancer
• Cancer cells both lose the
“brakes” (eg Rb and p53 tumor
suppressor genes) & often have
a “stuck gas pedal”
Key question – how can we target
this defect to selectively kill cancer
cells?
Once we do it in cells and mice, can
we design a human clinical trial…

14. Some more common techniques relevant to
cell biology I use now
Doing high-throughput drug screening MTT measures mitochondrial
for good drugs and combinations using reducing activity in the cell – is
MTT a surrogate for live cell number
LIVE CELLS
Yellow Purple insoluble
powder chemical (in
H2O)
Increasing dose of drug

15. Studying the cell cycle: What phase of the cell
cycle does a particular drug affect?
Flow cytometry – uses lasers to Cells are incubated with a fluorescent dye (propidium
capture information about single iodide) that binds to DNA then the machine measures
cells, size, shape, fluorescence etc how much fluorescence per cell
Basic Principle: Amount of DNA
S
G1 G2/M
(DNA
synthesis)
1X DNA Between 1- 2X DNA
content 2X DNA
content
content

16. Observations about how cells die
Our old friend, electron microscopy
Autophagy
Apoptosis
Mitotic
Necrosis catastrophe
If we know by what method(s) cells die, we can try to understand why!

17. More cell death – all about the nuclei!
Nuclear morphology as a measure of
apoptosis (one way cells can die) –
cells are stained with a fluorescent blue
dye (called DAPI)
TISSUE
NO TISSUE DAMAGE USUALLY
DAMAGE /INFLAMMATORY NO TISSUE
RESPONSE DAMAGE

18. Questions?
Email me @ thecancergeek@gmail.com or
talk to me on twitter @thecancergeek