2005_pset7_ans - 7.06 Problem Set #7, Spring 2005 1. a. You...

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7.06 Problem Set #7, Spring 2005 1. a. You are a grad student studying genetic alterations that lead to tumorigenesis. While plating primary cell cultures from a healthy person and a tumor from a cancer patient, you mix up the samples. A colleague suggests that you use the differences in growth properties between the two samples to distinguish them. Describe how the growth properties of cancer cells differ from those of healthy primary cells. Unlike normal primary cell cultures, cancer cells will grow indefinitely in culture. Normal primary cultures will stop dividing after several passages. (A passage is when cells are re- plated at low density to allow cells to grow and divide up to high cell density again.) Cancer cells also lack contact inhibition, and will grow on top of each other once the cells have divided enough times to form a confluent monolayer, unlike normal cells. In addition, cancer cells will grow in soft agar, unlike normal cells. Finally, cancer cells will often grow in media that does not contain any growth factors, while normal primary cell cultures have a strict dependence on growth factors. b. When studying the cancer cells from part a) , you discover that there is high Ras activity in the cells, but you can find no mutations in the Ras gene. You then start investigating different Receptor Tyrosine Kinases. You find by immunofluorescence, using a monoclonal antibody against the kinase domain, that one specific RTK is now located throughout the cytosol instead of in the plasma membrane. You then perform Western blot analysis on protein extracts from these cancer cells, and find that the RTK is much larger than normal. What kind of mutation in the RTK is indicated by these results, and how does that specific type of mutation lead to cancer? The chromosome containing the gene that encodes the RTK protein must have undergone a translocation with another chromosome, so that a chimeric gene was formed. A chimeric gene is a gene that is a fusion between two different normally occurring genes that are usually physically separated. In this case, a chimeric gene must have been formed between a gene that encodes a cytoplasmic protein and the gene that encodes the RTK. This chimeric gene, when transcribed and translated, now produces a chimeric protein whose N terminus is from a random cytoplasmic protein, and whose C terminus is from the kinase domain of the RTK. The protein fused to the kinase domain probably is a dimer (or some sort of oligomer), so that the kinase domains of the RTK are now constitutively dimerized, and therefore constitutively active, due to the constitutive dimerization of the N terminus of the chimeric protein. Constitutively active RTK would lead to constitutive activity of Ras, and thus the promotion of growth and division when it is inappropriate for the cells to grow and divide.
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c. In addition to differences in growth properties, cancer cells also have morphological differences, such as a disorganized cytoskeleton. The Rho family of small GTPases is
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2005_pset7_ans - 7.06 Problem Set #7, Spring 2005 1. a. You...

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