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DNA Content / Cell Cycle Analysis. T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division Beckman Coulter, Inc Miami, FL Vincent.Shankey@Coulter.com. Propidium Iodide Fluorescence Bound to DNA. DNA Content and Cell Cycle Analysis. CB Bagwell Verity Software House. Mitosis.
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DNA Content / Cell Cycle Analysis T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division Beckman Coulter, Inc Miami, FL Vincent.Shankey@Coulter.com
Propidium Iodide Fluorescence Bound to DNA DNA Content and Cell Cycle Analysis CB Bagwell Verity Software House
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
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)
Mitosis Redefined-Transition States From Pines & Reider, Nature Cell Biol 3:E3-6, 2002
The Cell Cycle as a Finite State Machine Finite State Machine (A or B) Digital System James Jacobberger, CWRU
Finite States of the Cell Cycle James Jacobberger CWRU
G2/M Transition/Finite States defined by Flow Cytometry (D) Anaphase Telophase P-H3 Alexa 647 Metaphase Cytokenesis G2 Prometaphase Cyclin A2 PE
Finite States Protein synthesis and degradation (Cyclins) and activation (kinases) makes the cell cycle “one way”
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)