General Path• Bachelors degree (~4yrs) in a science

• (Only sometimes) Masters degree 1-2yrs, classes & perform focused research project

• PhD degree, some classes and full-time research (have to discover something NEW!), usually paid for RA/TA, 5-7+yrs, write a 100-200 page dissertation at end

• Postdoctoral fellowship(s) 2-5+ yrs – full time research, learning how to write grants, more research training, how to run a lab etc

What I Did• Bachelors degree (in Finance) but

as part of a individually developed curriculum took enough biology and chemistry to get 2 minors

• PhD in Molecular Carcinogenesis at the UTHSC/MDACC, Smithville TX

• TRIUMPH postdoctoral fellow, at the University of Texas MD Anderson Cancer Center, Houston TX

What education/training after high school does it take to

become a (life) scientist?

2000-2004

2004-2011

2011-now

Education in pictures

Smithville (1hr from

Austin), TX Houston, TX

Newark DE

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!

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)!

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!

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

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

Techniques in a cell biology lab (cont.)

Fluorescence and confocal microscopy – use this to observe

protein localization (what organelles are important?)

Supplementary Figure S3

Flag-TSC2

PEX5IP: PEX5

IgG WT RQ RW RG

e

a

HEK 293 cells

IP: Myc

Input

Flag-TSC2

Flag-TSC2

Myc-TSC1

Myc-TSC1

IgG WT RQ RG RW

L1624P

G294E

HEK 293 cells

IP: Flag

PEX5 (light)

Flag-TSC2

IgG WT RQ RW RG

PEX5 (dark)

HEK 293 cellsb

c

WT

PMP70 Merge/DAPI

RQ

PMP70 Merge/DAPI

RG

PMP70 Merge/DAPI

RW

PMP70 Merge/DAPI

HeL

a c

ells

Flag

Flag

Flag

Flag

d

Calnexin Merge/DAPI

RQ

RQ

HeL

a c

ells

Flag

VDAC Merge/DAPIFlag

More cell biology techniques

Electron microscopy – observe structural

information inside cells without using

antibodies to detect individual proteins

Animal/human tissue studies

– immunohistochemistry

staining for proteins using

antibodies then chemical

reactions that generate

brown color

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.

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…

Some more common techniques relevant to

cell biology I use now

Doing high-throughput drug screening

for good drugs and combinations using

MTT

MTT measures mitochondrial

reducing activity in the cell – is

a surrogate for live cell number

Yellow

powder

Purple insoluble

chemical (in

H2O)

Increasing dose of drug

LIVE CELLS

Studying the cell cycle: What phase of the cell

cycle does a particular drug affect?

Flow cytometry – uses lasers to

capture information about single

cells, size, shape, fluorescence etc

Amount of DNA

Cells are incubated with a fluorescent dye (propidium

iodide) that binds to DNA then the machine measures

how much fluorescence per cell

Basic Principle:

G1S

(DNA

synthesis)

G2/M

2X DNA

content

1X DNA

content

Between 1-

2X DNA

content

Observations about how cells die

Autophagy

Mitotic

catastrophe

Apoptosis

Necrosis

Our old friend, electron microscopy

If we know by what method(s) cells die, we can try to understand why!

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

DAMAGE

/INFLAMMATORY

RESPONSE

NO TISSUE

DAMAGE

USUALLY

NO TISSUE

DAMAGE

Questions?

Email me @ thecancergeek@gmail.com or

talk to me on twitter @thecancergeek