Decrease in actin gene expression in melanoma cells compared to melanocytes is partly counteracted by BrdU-induced cell

adhesion and antagonized by L-tyrosine induction of terminal differentiation.

Gomez LA, Rieber MS, Rieber M. 995Biochem and Biophys Res Comm 216:84-89

Give full reference information

Bob Smith and Alicia ApplegateFall 2011

Purpose of study

What factors are important to the metastasis of melanoma cells

Key Question

Do the effects of BrdU on cell shape involve changes to the actin cytoskeleton

Purpose: is the broadest reason for performing the investigation

Key Question: specific topic of the investigation

Experimental system

• Used normal mouse melanocytes and B16 melanoma cells

• Cultured

• Studied effects of BruU and/or tyrosine on actin gene expression

Description of the experimental system is broad and generally applies to all experiments in the study

Why do researchers study effects of BrdU?

• Bromodeoxyuridine (BrdU) • Thymidine nucleoside analog• Causes mutations

– Induced by UV radiation

• Previous Studies have shown BrdU– Effects cell adhesion– Change melanocyte shape and invasiveness– Potential treatment?

Give enough background information for your audience to understand key terms

and concepts

Why is L-Tyrosine studied?• Amino Acid

• Involved in melanin pigment synthesis- Characteristic of melanocytes

• Induces differentiation of melanoma cells

pdrhealth.com

Supplement with relevant graphics whenever possible

In the following presentation of different experimentseach section includes the relevant

Methodology, Results, and Interpretations

Avoid unnecessarily using slide titles like “Procedures”“Results”

“Description” (especially big ugly titles that take up an

entire slide or a large portion of one)

Introduce each experiment with the specific question it seeks to answer

It is generally obvious what will be procedures, results etc.

• To cultures of B16 melanoma cells were added:– BrdU, L-tyrosine, or BrdU + L-tyrosine

• Treated cultures with trypsin to cause detachment

How did BrdU and tyrosine affect cell attachment?

No need for “Procedures” label – you can simply state it.

Results

Explanation:

Tyrosine believed to cause differentiation

of cells, reducing BrdU incorporation

1) % attachment

reduced by BrdU

2) Effect inhibited or reversed by tyrosine

Combine Description of Results and Interpreations on same slide (whenever possible)

State key results (above), and point out data in Figure to the audience as you do so.

Explanations of results are essential.

• BrdU, L-tyrosine or BrdU + L-tyrosine treated B16• Compared to normal melanocytes

– BrdU, L-tyrosine or BrdU + L-tyrosine• After 5 days

– total RNA extracted – Ethidium bromide staining (total RNA)

Actin mRNA– Actin mRNA identified with actin probe– Labeled with P32 – Northern blot performed– Autoradiaography and densitometry

How did BrdU and tyrosine affect actin gene expression?

You must be very judicious in the level of detail included with procedures

Include what is necessary for your audience to understand the results

You can verbally add procedural details

Results

EtBr stained actin mRNA

C = untreated B16

mel = normal melanocytes

Notice that Figures do not have lengthy legends, as would be

found in original article

You may need to supply a key to abbreviations (as above), but you should talk through rest of figure

details

Quantification of mRNA expression dataNorthern blot subject to

densitometry

Results-- BrdU increased actin

mRNA

-- Tyr or BrdU + Tyr similar to control

-- not as much mRNA as in normal cells

mel

BrdU

Explanation

• Effect of BrdU is to increased actin mRNA expression in B16

• Reversed again by Tyr induced differentiation

Notice additional labels added to this

figure for clarity

How does UV light exposure effect actin mRNA expression?

• BrdU treated cells exposed to UV light

• Relative expression of actin mRNA

• GAPDH mRNA used as a control

ResultsUV light treatment Increased the amount of actin expressed in the BrdU cells vs. the BrdU/UV cells.

Explanation

BrdU treated cells are more sensitive to UV light induced damage

Overall conclusions Effect of BrdU• BrdU affects actin cytoskeleton• BrdU increases actin gene expression• UV light affect

Effect of tyrosine • cell differentiation• less cell division• less BrdU incorporation

Thus, changes in gene expression may be important factor in melanoma metastasis

The overall conclusions summarize findings for all

experiments

And indicate how those results relate back to broad

purpose of the article

Critique of Investigation• Cell attachment methodology and

measurements not clearly explained• Which type of actin is probed?• No statistical analysis performed• Medical significance of UV treatment is unclear• BrdU is a mutagen; thus seems unlikely

candidate as drugCritique of the article reflects how well you really understand it.

Avoid shallow critiques like:“It was confusing”“Not much background was given about…”“They did not explain their results…”

Overall Conclusions

• BrdU exposed to UV light decreases actin expression

• BrdU increases actin expression and cellular adhesion

• L-tyrosine with BrdU decreases actin expression

• Conclusions support idea that actin gene involved in melanoma growth.