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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