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DNA Content / Cell Cycle Analysis

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  1. DNA Content / Cell Cycle Analysis T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division Beckman Coulter, Inc Miami, FL Vincent.Shankey@Coulter.com

  2. Propidium Iodide Fluorescence Bound to DNA DNA Content and Cell Cycle Analysis CB Bagwell Verity Software House

  3. Mitosis Prometaphase Metaphase Telophase Anaphase Prophase Cytokinesis G0 G1 S G2 DNA Content, Phase Fractions, and Time Time in Cell Cycle Phase (Hrs) 8 hrs 2 hrs 0.5 hr 4 – 40 hrs James Jacobberger CWRU

  4. Mitotic Cell Cycle

  5. P-H3 Alexa 647 Cyclin A2 PE Gate Percent of G2/M Events G 36% (includes G2) H 0.24% I 1.40% J 0.32% K 0.35% L 0.35% M 27% (includes late S)

  6. Mitosis Redefined-Transition States From Pines & Reider, Nature Cell Biol 3:E3-6, 2002

  7. The Cell Cycle as a Finite State Machine Finite State Machine (A or B) Digital System James Jacobberger, CWRU

  8. Finite States of the Cell Cycle James Jacobberger CWRU

  9. G2/M Transition/Finite States defined by Flow Cytometry (D) Anaphase Telophase P-H3 Alexa 647 Metaphase Cytokenesis G2 Prometaphase Cyclin A2 PE

  10. J. Jacobberger, CWRU

  11. Finite States Protein synthesis and degradation (Cyclins) and activation (kinases) makes the cell cycle “one way”

  12. Why We Need Guidelines

  13. ConclusionsClinical Application of DNA Content/S-Phase • Many studies (published from ~1975-1995) correlated DNA ploidy or S-phase (rare) with tumor grade or stage • Few correlated with outcome – the real endpoint! • Poor quality of data limited the potential impact of many published studies • The DNA Consensus Guidelines came too late! • Bagwell, et al study demonstrated the clinical impact of DNA content/S-phase measurements • Too late! (published after the ASCO/JNCI consensus on biomarkers for breast and colorectal Ca)