Pfeffer 1

lpfeffer@utmem.edu

Overview
70. Overview of Cell Cycle
To understand:
General Cell Cycle control
Phases of cell cycle
Experimental systems used to study cell cycle
Regeneration potential of different cells
Role of checkpoints
How to determine length of cell cycle and its phases
Mitosis versus meiosis
Test questions directly off poweroints and objectives.
All powerpoints on web.
This list is on blackboard separately. Pfeffer directs new cancer building.
2
Eukaryotic cell cycle 4 phases: G1, G2, M,S.
Cell cycle controlled by discrete regulatory pathways. Orchestrated carefully with checkpoints where cell can sense if something is not right and arrest or die.
Central point of control is cyclically activated cyclin-dependent kinases.
Last 5 years 2 sets of Nobel prizes were awarded for discovery of cell cycle control and ubiquitination pathway.
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Typical mitosis in mammalian cell takes 1 hour. Human cell cycle 24 hours. M phase is linear series of events to get cell out of mitosis. Deformation- checkpoint stops cells before telophase in somatic cells. Best understood part of cell cycle is mitosis. Cytokinesis is dividing cytoplasm. Cytokinesis is the only part of mitosis that is not exact. One cell may get a bit more or less cytoplasm than the other. Gap phases provide time for monitoring signals to prepare for next phase. G1- all preparation for S completed. G2- all proteins for M manufactured except for cyclin.
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DNA is only synthesized during S phase.
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pre 1950 could only see mitosis and interphase under microsope.
Swift found discrete part of cell cycle when DNA was replicated.
H and P named S phase for synthesis, and G1 and G2 for gaps in cycle.
Xenopus laevis is African clawed toad.
Kinds of things controlling cell cycle in organisms from surf clam to yeast to frog are same.
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Xenopus oocyte is big.
Fibroblasts are used in culture because they grow well (lower picture). Piece of skin in plastic Petri dish wth serum and media- they grow easily.
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Duration of cell cycle listed here.
Amount of DNA listed here too. 1n haploid, 2n diploid, 4n tetraploid.
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Why frog eggs?
Enormous- can see with naked eye. Egg has million times amount cytoplasm as fully mature frog cell. Easy to inject something into it. Oocyte G1 and G2 done away with. Has eveything in cytoplasm ready to divide. MPF is maturation or mitosis promoting factor. It is a complex sufficient to drive cell from G2 into mitosis.
During fertilization- pseudohaploid polar body becomes halploid cell, which mixes with 1n from sperm to make diploid cell.
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Mammalian cells a lot smaller. Grow well. MPF from frog can be used to drive fibroblasts into mitosis.
Mutant cell lines with defects have been used to study cell cycle control. G1/S and G2/M transition studied and well understood. Bromodeoxyuridine looks like thymidine.
Flow cytometer- get pattern on left.
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3 kinds of cells in body listed here.
Permanent are not thought to regenerate.
Stable- hepatocytes. In rat liver you can cut out 2 out of 3 lobules. Regenerate in one week.
Labile cells continuously divide.
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To enter M phase the cell must assemble a mitotic spindle. Get out- anaphase and cytokinesis. Checkpoints located G1/S, G2/M.
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Checkpoint questions. Cell in low serum, goes into G0. Nutrients are not there, so cell slows down.
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Cell mass increases to critical point, chromosomes segregate, cell division happens.
M phase cyclins just made during mitosis.
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Classic experiment. Fused cells in different parts of cycle.
Something in S phase cell can drive DNA synthesis for G1 cell, but not G2. Cell must be competent for signal. There is a soluble factor to tell G1 to go into S. Something in M cell drives other cells into M phase.
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G1 critical part cell cycle. If cell not ready, goes into sleep or G0 phase.
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Studies in late 1950s and 60s by Hayflick. Lifespan of human fibroblasts is closely regulated. Newborn- 50 -60 doublings. Older 15-20. There is an inverse relationship between age and proliferation potential of fibroblasts.
Progeria- something limits lifespan of cells.
Telomeres shorten as you age. One way to immortalize cells- express telomerase gene.
Senescence is a specific cell state in which protein and DNA synthesis slow down and proliferation potential ends.
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Cell labelling
Radioactively labelled thymidine
BrdU
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Feed cells label, take cells out, figure out # labels
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Percent labelled mitosis: Only S phase cells are labelled.
26-27
Graph generated in experiment.
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Shows doubling time 20 hours, not 22.
29 Cancers can grow slow or fast, but they usually have similar doubling time to counterpart in normal cells. They just do not know when to stop.
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Flow cytometry is easier.
Label with DNA dye.

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Fluorescent detector
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Different tumors have varying doubling times.
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Mitotic cells change shape.
One way to get synchronized cells- 4% of cells at any one time are undergoing mitosis. Telandek (this is phonetic spelling)- grow fibroblasts on roller bottle with tubing that sprayed media- shake- all mitotic cells came off. 95% pure.
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Start seeing mitotic spindle.
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Metaphase plate with chromosomes lined up in middle.
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Mitosis vs meiosis.
Somatic cells undergo mitosis, gametes meiosis.
Meiosis one produces pseudodiploid cell. Second meiotic division yields 1n amount of DNA.
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platelet derived growth factor was one of first.
50-51
Hydroxyurea blocks cells going into DNA synthesis and causes cell death. All cells in S phase die. Remove and wash- cells march synchronouslty thru cell cycle.