The Big Idea
Over the last 28 years many defects in genes have been linked to cancer, each promising
to be the magic in understanding and curing cancer. The understanding now indicates
cancer as a multistep process, each of these steps generally due to a genetic aberration.
Accumulation of these mutations in genes allows the cell to progress to tumor and
malignancy.
Every cancer can be attributed to a different set of genetic aberrations, and different
genes are either expressed or not expressed. More than 100 different types of cancer can
be found within specific organs. Each caner has a different potential of being treated by
current therapies. For example, it has been shown cancer cells that lack the p53 protein
do not respond well to radiation therapy, and other non-malignant cells lacking p53 will
readily progress to malignancy in response to radiation. Thus the treatment itself causes
more cancers.
The best way to treat a cancer then would be to know which genes are mutated and which
genes are expressed or not expressed in the tissue. One approach that would allow you to
look at numerous genes expressed and use that knowledge to determine treatment. Gene
expression of numerous genes can be looked at by a new technique called microarray
analysis.
Some questions to think about before the activity:
How does one determine the function of a gene?
Which genetic aberrations have been implicated in cancer?
What cellular functions are affected (turned on or off) in cancer cells, and how might
these affect normal cell development?
Microarray Technology:
Microarray technology can place all of the genes that have been sequenced for an
organism and by simple hybridization ask which of the genes are producing mRNA (and
therefore expressed) in specific cells or tissues. The DNA of the sequenced genes is
"spotted" onto a microscope slide. Up to 20,000 genes can be analyzed at one time, but
for this activity only 50 genes have been spotted for your analysis. Messenger RNA was
extracted from breast tissue that was biopsied from a cancer. This will be compared to
breast tissue that did not contain the caner. All of the mRNA from each of the tissues
was reverse transcribed into cDNA. The mRNA from the normally breast tissue was
transcribed into DNA with a blue dye label, whereas the mRNA from the breast cancer
tissue was transcribed into DNA with a red label. These cDNAs can be mixed together
and applied to the microarray slide that has been adhered with genes of cellular processes
often found aberrant in breast cancers.
Before beginning this activity be sure to watch an animation on microarrays using yeast
grown under the conditions of with and without oxygen.
http://occawlonline.pearsoned.com/bookbind/pubbooks/bc_mcampbell_genomics_1/medi
alib/method/chip/chip.html
Materials for the activity:
 Microarray slide -- this slide contains genes involved in cancer
 Disposable pipette
 cDNA mixture solution -- these cDNAs were made from normal breast tissue
(attached to blue dye) and breast cancer tissues (attached to red dye).
 Wash solution
 Color developing reagent
You will look develop the microarray and then analyze which genes were "expressed"
under which conditions.
Procedure:
Get a microarray slide, a disposable pipet, a tube labeled cDNA and a paper towel.
1. Place the slide onto the paper towel.
2. Add enough of the cDNA solution to the slide to completely cover it, but not spill
off of the slide.
3. Let the cDNA hybridize with the microarray slide for 5 minutes.
4. After the 5 minute incubation of the microarray slide with cDNA, rinse off the
excess cDNA with the microarray wash solution (in squeeze bottle).
5. Add color solution, again enough to cover the slide but not spill over the slide.
This solution is toxic so take care to not get it on you, and wash off of skin
immediately. Let the color solution set for 30 sec, then wash off excess with
microarray wash solution.
6. Record you data.
Draw your results of the microarray:
Which genes were expressed in cancer tissue? Which genes were expressed only in the
cancer tissue?
Color in the spots in the above pictorial representation of a slide with the appropriate
corresponding color that you see on your slide.
Which genes are not expressed cancer cells? Why would some genes not be turned on in
cancer tissue? Can you think of any other conditions that you might see a difference in
genes being expressed? Please list or discuss.
List of cancer related genes that were spotted on the microarray slide:
Symbol
POL1
GAPdH
HK1
Name
DNA Polymerase
Glyc.Ald.Phos.DeH-ase
Hexokinase 1
ALDH1
GLUT1
ACTG1
DNASE1
RNASE4
TOP1
BRCA1
PDGFR
CYP1A1
BCL2
LIG1
POL1
AldDeHase
Glucose Transporter 1
Actin, cytoplasmic
Deoxyribonuclease I
Ribonuclease 4
Topoisomerase I
Breast cancer type 1 susceptibility protein
Platelet-derived Growth Factor Receptor
Cytochrome P450 1A1
B-cell lymphoma protein 2
DNA Ligase I
DNA Polymerase Iota
APAF1
p53
ZNF84
MUC1
G6PD
Apoptosis Protease Activating Factor 1
p53 (tumor protein 53)
Zinc Finger Protein 84
Transmembrane Mucin 1
Glu.6-phosp DeH-ase
Function
DNA replication
Kreb Cycle
Glycolysis
Converts retinal to retinoic acid, overexpression confers
cyclophos. resistance.
Transports glucose molecules into cells for energy
Microtubule formation, cytoskeleton formation
Degrades DNA
Degrades RNA
Aids in DNA supercoiling
Plays a role in DNA double-strand break repair
Integral membrane receptor that binds PDGF
Drug metabolism
Supresses apoptosis
DNA Ligation during replication/repair
Synthesizes DNA on a template strand
Tumor suppressor- Promotes apoptosis in damaged/ irregular
cells
Tumor supressor- induces growth arrest and/or apoptosis
May play a role in transcription regulation
Plays a role in cell adhesion, cell to cell interactions
Metabolism, Provides pentose sugars for nucleic acid synth.
TNF
ADH4
Tumor necrosis factor
Alcohol Dehydrogenase
DNMT1
POLR2A
MDM2
MMP3
VEGF
ACAT1
MCR4
PDK2
GPB
DUSP1
PRL1
DNA Methyltranferase I
RNA Polymerase, subunit 2
MDM2
Matrix Metalloprotease 3 (Stromelysin)
Vascular endothelial growth factor
Acetoacetyl-CoA thiolase
Melanocortin receptor
Pyruvate DeH-ase Kinase
Glycerol Phosphatase Beta
Dual-specificity protein 1
Protein Tyrosine Phosphatase
JUN
Jun
FOS
RASSF1
RAS
Fos
Ras-association domain, family 1 protein
ras
SOS
EGFR
CS
AChE
CDKNA
IL6
GSTP1
VEGFR
sos
Epithelial Growth Factor Receptor
Citrate Synthase
Acetylcholinesterase
p21
Interleukin 6
Glutathione S-transferase
Vascular Endothelial Growth Factor Rec.
PLCG1
MYC
RPS18
NAT1
MAPK
Phospholipase-C gamma
c-Myc Proto-oncogene
Ribosome Subunit 18S
n-acetyltransferase
Mitogen-activated Protein Kinase
Cytokine, may induce tumor cell death. Deficiencies common
in cancer
Alcohol processing
Modifies DNA to make it inaccessible thereby inhibiting
transcription
RNA Polymerase synthesizes RNA
Inhibits p53-induced arrest and cell death
Degrades extracellular matrix that anchors cells in place
Growth factor that promotes formation of new blood vessels
Ketone body metabolism
Binds melanocortin; multiple downstream effects
Phosphorylates/inhibits PDH complex
Inhibits glycogen phosphorylase
Dephosphorylates and "resets" MAPK
Stops growth signal cascade from receptor tyrosine kinases
Component of AP-1 transcription factor- activates
transcription
Component of AP-1 transcription factor- activates
transcription
Inhibits cell cycle progression at the G1-S phase transition
Small G-protein, signaling molecule in transcription activation
Tyrosine-kinase receptor signaling molecule, binds SH3
domains
Binds EGF to promote epithelial cell growth
Kreb Cycle enzyme
Degrades Ach to stop action potential in nerves
Works with p53 to stop cell cycle progression
Cytokine, differentiation of b-cells, nerve cells
Helps to inactivate and eliminate some types of toxins
Binds VEGF, promotes growth of new vasculature
Cleaves Phosphatidyl Inositol TriPhosphate into IP3 and DAG
for signaling
Activates transcription of growth-related genes
Ribosomes translate mRNA into protein
Modifies histones,
Signaling molecule and transcriptional activator