UNC-Duke Biology Course for Residents Fall 2018 1 Cell Cycle: Sequence of changes in a cell starting with the moment the cell is created by cell division, continuing through the doubling of the DNA and other cellular contents, and ending with cell division into 2 daughter cells Phases of the cell cycle: I. Interphase (G1, S, and G2): the part of the cell cycle that excludes mitosis G1: Gap between previous cell division (M) and DNA replication (S) S: DNA replication G2: Gap between DNA replication (S) and cell division (M) II. Mitosis (M): where copied DNA and cytoplasm are divided between two daughter cells Cells can exist in a quiescent state (G0) where they are not in an active cell cycle. The decision to enter into an active cell cycle depends on extra-cellular signals and nutrients levels that signal to the nucleus. A complex molecular circuitry ( The Cell Cycle Clock ) integrates all of these signals to decide whether to enter into an active cell cycle. 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 1
UNC-Duke Biology Course for Residents Fall 2018 2 Red = oncogenes or tumor suppressor genes 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 2
UNC-Duke Biology Course for Residents Fall 2018 3 Mitosis can be subdivided into prophase, metaphase, anaphase, and telophase: Prophase: Chromosomes condense and become visible under microscope. Centrosomes assemble at opposite poles and bind to microtubules that will form the mitotic spindle. Metaphase: Chromosomes align in a symmetrical fashion as they become attached at the mitotic spindle. Nuclear membrane disappears. Anaphase: The two copies of the DNA are pulled into opposite directions. Telophase: Chromosomes de-condense and a nuclear membrane forms around each set of chromosomes. The cell completes division into 2 daughter cells. Prophase Metaphase Anaphase Telophase Blue = chromosomes Green = microtubule fibers of the mitotic spindle Centrosome: is made up of 2 centrioles that are each composed of 9 triplets of microtubules 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 3
The Cell Cycle Through the Ages UNC-Duke Biology Course for Residents Fall 2018 4 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 4
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UNC-Duke Biology Course for Residents Fall 2018 7 Cell Cycle and Differentiation of Normal Tissue Stem Cells Normal tissue stem cells have 2 defining properties: (1) self-renewal (2) differentiation Cell Cycle and Differentiation of Cancer Stem Cells Clonal Evolution Model Cancer Stem Cell Model Cancers can follow the clonal evolution model or the cancer stem cell model (or may be a mix of the two models). Emerging evidence suggests that cancer stem cells may be resistant to radiation. Reya et al Nature 2001 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 7
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UNC-Duke Biology Course for Residents Fall 2018 9 Interphase Cell Mitotic Cell 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 9
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UNC-Duke Biology Course for Residents Fall 2018 15 Zeman and Bedford Radiation Res 101: 373-393, 1985 The Modern-Day Cell Cycle Cell cycle checkpoints: Control mechanisms to ensure that the next step in the cell cycle does not proceed until the specific molecular events have occurred to complete the current phase of the cell cycle. Many of the cell cycle checkpoints are related to the status of DNA (DNA damage) and can be triggered by radiation. 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 15
To move from one phase of the cell cycle to the next, proteins are phosphorylated by a complex of a cyclin and a cyclin-dependent kinase (CDK): G1: Cyclin D-CDK4 or CDK6 Cyclin E-CDK2 S: Cyclin A-CDK2 Cyclin A-CDC2 M: Cyclin B-CDC2 UNC-Duke Biology Course for Residents Fall 2018 16 Levels of cyclins change across the cell cycle How does radiation cause cell cycle arrest? CDK Inhibitors are up-regulated by radiation and activate cell cycle checkpoints. 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 16
G1 Checkpoint: Prevents S phase entry until after DSB is repaired Radiation activates ATM and CHK2 protein kinases that phosphorylate p53 protein and increase p53 protein levels. p53 increases p21 levels that block cycline-cdk. UNC-Duke Biology Course for Residents Fall 2018 17 CDKs are regulated not only by the levels of cyclins and CDKIs, but also by the phosphorylation status of the CDK. G1 Checkpoint: For CDK2 to be active, CDK2 must also have an inhibitory phosphate removed by the Cdc25A phosphatase. CHK2 phosphorylates Cdc25A and targets it for degradation. Without CDC25A, CDK2 retains an inactivating phosphorylation. 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 17
UNC-Duke Biology Course for Residents Fall 2018 18 CDKs are regulated by the phosphorylation status of the CDK to control entry into mitosis. Passage through mitosis requires high levels of cyclin B and active Cdc2. For Cdc2 to be active, specific amino acids must not have phosphate groups. This adds another layer of regulation with phosphatases (like Cdc25c) that remove phosphate groups to activate Cdc2 and kinases (like Wee1) that add phosphate groups to inactivate Cdc2. The phosphorylation of CDKs can change after radiation, which is another way that radiation can alter cell cycle progression. Question: Why do most tumor cells not have a G1 checkpoint after radiation? Summary: 1) Cell cycle progression depends on the activity of cyclin-cdk complexes a) Cyclin levels change in different phases of the cell cycle b) CDKs are also regulated by phosphorylation c) CDKI s block cyclin-cdk activity 2) DNA damage response to radiation can alter CDK activity through these layers of regulation 10/31/18 David Kirsch, M.D., Ph.D., and Elaine Zeman, Ph.D. 18