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Diagnosis of Breast Caner Tissue with Microarray
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 genetic aberrations. 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?
How is cDNA made and used in microarray technology?
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/medialib/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 pipette, a tube labeled cDNA and a paper towel.
- Place the slide onto the paper towel.
- Add enough of the cDNA solution to the slide to completely cover it, but not spill off of the slide.
- Let the cDNA hybridize with the microarray slide for 5 minutes.
- After the 5 minute incubation of the microarray slide with cDNA, rinse off the excess cDNA with the microarray wash solution (in squeeze bottle).
- 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.
- 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:
| 1) | Symbol | Name | Function |
| 2) | POL1 | DNA Polymerase | DNA replication |
| 3) | GAPdH | Glyc.Ald.Phos.DeH-ase | Kreb Cycle |
| 4) | HK1 | Hexokinase 1 | Glycolysis |
| 5) | ALDH1 | AldDeHase | Converts retinal to retinoic acid, overexpression confers cyclophos. resistance. |
| 6) | GLUT1 | Glucose Transporter 1 | Transports glucose molecules into cells for energy |
| 7) | ACTG1 | Actin, cytoplasmic | Microtubule formation, cytoskeleton formation |
| 8) | DNASE1 | Deoxyribonuclease I | Degrades DNA |
| 9) | RNASE4 | Ribonuclease 4 | Degrades RNA |
| 10) | TOP1 | Topoisomerase I | Aids in DNA supercoiling |
| 11) | BRCA1 | Breast cancer type 1 susceptibility protein | Plays a role in DNA double-strand break repair |
| 12) | PDGFR | Platelet-derived Growth Factor Receptor | Integral membrane receptor that binds PDGF |
| 13) | CYP1A1 | Cytochrome P450 1A1 | Drug metabolism |
| 14) | BCL2 | B-cell lymphoma protein 2 | Supresses apoptosis |
| 15) | LIG1 | DNA Ligase I | DNA Ligation during replication/repair |
| 16) | POL1 | DNA Polymerase Iota | Synthesizes DNA on a template strand |
| 17) | APAF1 | Apoptosis Protease Activating Factor 1 | Tumor suppressor- Promotes apoptosis in damaged/ irregular cells |
| 18) | p53 | p53 (tumor protein 53) | Tumor supressor- induces growth arrest and/or apoptosis |
| 19) | ZNF84 | Zinc Finger Protein 84 | May play a role in transcription regulation |
| 20) | MUC1 | Transmembrane Mucin 1 | Plays a role in cell adhesion, cell to cell interactions |
| 21) | G6PD | Glu.6-phosp DeH-ase | Metabolism, Provides pentose sugars for nucleic acid synth. |
| 22) | TNF | Tumor necrosis factor | Cytokine, may induce tumor cell death. Deficiencies common in cancer |
| 23) | ADH4 | Alcohol Dehydrogenase | Alcohol processing |
| 24) | DNMT1 | DNA Methyltranferase I | Modifies DNA to make it inaccessible thereby inhibiting transcription |
| 25) | POLR2A | RNA Polymerase, subunit 2 | RNA Polymerase synthesizes RNA |
| 26) | MDM2 | MDM2 | Inhibits p53-induced arrest and cell death |
| 27) | MMP3 | Matrix Metalloprotease 3 (Stromelysin) | Degrades extracellular matrix that anchors cells in place |
| 28) | VEGF | Vascular endothelial growth factor | Growth factor that promotes formation of new blood vessels |
| 29) | ACAT1 | Acetoacetyl-CoA thiolase | Ketone body metabolism |
| 30) | MCR4 | Melanocortin receptor | Binds melanocortin; multiple downstream effects |
| 31) | PDK2 | Pyruvate DeH-ase Kinase | Phosphorylates/inhibits PDH complex |
| 32) | GPB | Glycerol Phosphatase Beta | Inhibits glycogen phosphorylase |
| 33) | DUSP1 | Dual-specificity protein 1 | Dephosphorylates and "resets" MAPK |
| 34) | PRL1 | Protein Tyrosine Phosphatase | Stops growth signal cascade from receptor tyrosine kinases |
| 35) | JUN | Jun | Component of AP-1 transcription factor- activates transcription |
| 36) | FOS | Fos | Component of AP-1 transcription factor- activates transcription |
| 37) | RASSF1 | Ras-association domain, family 1 protein | Inhibits cell cycle progression at the G1-S phase transition |
| 38) | RAS | ras | Small G-protein, signaling molecule in transcription activation |
| 39) | SOS | sos | Tyrosine-kinase receptor signaling molecule, binds SH3 domains |
| 40) | EGFR | Epithelial Growth Factor Receptor | Binds EGF to promote epithelial cell growth |
| 41) | CS | Citrate Synthase | Kreb Cycle enzyme |
| 42) | AChE | Acetylcholinesterase | Degrades Ach to stop action potential in nerves |
| 43) | CDKN1A | p21 | Works with p53 to stop cell cycle progression |
| 44) | IL6 | Interleukin 6 | Cytokine, differentiation of b-cells, nerve cells |
| 45) | GSTP1 | Glutathione S-transferase | Helps to inactivate and eliminate some types of toxins |
| 46) | VEGFR | Vascular Endothelial Growth Factor Rec. | Binds VEGF, promotes growth of new vasculature |
| 47) | PLCG1 | Phospholipase-C gamma | Cleaves Phosphatidyl Inositol TriPhosphate into IP3 and DAG for signaling |
| 48) | MYC | c-Myc Proto-oncogene | Activates transcription of growth-related genes |
| 49) | RPS18 | Ribosome Subunit 18S | Ribosomes translate mRNA into protein |
| 50) | NAT1 | n-acetyltransferase | Modifies histones, |
| 51) | MAPK | Mitogen-activated Protein Kinase | Signaling molecule and transcriptional activator |
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BIOTECH Project Last Modified March 1, 2002 |