Cell Cycle I Molecular Cell Biology November 6, 2014 Stephen Oh, M.D., Ph.D. Assistant Professor Division of Hematology

Outline Overview of the cell cycle Cell cycle regulation – fundamental concepts Cancer as a fundamental disruption in cell cycle regulation

What is the basic function of the cell cycle? Accurately duplicate the vast amount of DNA in chromosomes Segregate the copies precisely into genetically identical daughter cells Figure 17-2 Molecular Biology of the Cell, 4th Edition

The phases of the cell cycle Figure Molecular Biology of the Cell, 4th Edition G1 – gap between M and S phases S – DNA replication G2 – gap between S and M phases M - mitosis Interphase ~23 hours M phase ~1 hour Why are gap phases needed?

What critical features are needed for proper guidance through the cell cycle? Figure Molecular Biology of the Cell, 4th Edition

A clock, or timer, that turns on each event at a specific time A mechanism for initiating events in the correct order A mechanism to ensure that each event is triggered only once per cycle Binary (on/off) switches that trigger events in a complete, irreversible fashion Backup mechanisms to ensure that the cycle can work properly even when parts of the system malfunction Adaptability so that the system's behavior can be modified to suit specific cell types or environmental conditions Figure Molecular Biology of the Cell, 4th Edition What critical features are needed for proper guidance through the cell cycle?

The cell cycle is primarily regulated by cyclically activated protein kinases Figure 17-15, Molecular Biology of the Cell, 4th Edition

Malumbres M, Nature Reviews Cancer 2009 Evolution of cell cycle control: from yeast to humans

Table Molecular Biology of the Cell, 4th Edition Overview of major cyclins and Cdks of vertebrates and yeast

Bardin AJ, Nature Rev Mol Cell Biol 2001 Overview of major cyclins and Cdks of vertebrates and yeast

Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins Figure 17-18, Molecular Biology of the Cell, 4th Edition Why is cell cycle progression governed primarily by inhibitory regulation?

Figure Molecular Biology of the Cell, 4th Edition Cell cycle control depends on cyclical proteolysis

Mechanisms controlling S-phase initiation Figure Molecular Biology of the Cell, 4th Edition

DNA damage leads to cell cycle arrest in G1 Figure Molecular Biology of the Cell, 4th Edition

Figure Molecular Biology of the Cell, 4th Edition Overview of the cell cycle control system

Table Molecular Biology of the Cell, 4th Edition Summary of major cell cycle regulatory proteins

Figure Molecular Biology of the Cell, 4th Edition Mitogens stimulate cell division

Figure Molecular Biology of the Cell, 4th Edition Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death

Figure Molecular Biology of the Cell, 4th Edition Extracellular Growth Factors Stimulate Cell Growth

Figure Molecular Biology of the Cell, 4th Edition Extracellular Survival Factors Suppress Apoptosis

Intracellular signaling networks related to cell proliferation and cancer Hanahan and Weinberg, Cell 2011

Primary myelofibrosis Essential thrombocythemia Polycythemia vera JAK2 V617F Myeloproliferative neoplasms are clonal disorders derived from hematopoietic stem/progenitor cells

Proliferation/Survival JAK2 STAT3/5 P P P P P P P P P P P P TPO G-CSF JAK2 V617F JAK2 V617F STAT3/5 P P P P JAK-STAT activation is a hallmark of myeloproliferative neoplasms

Proliferation/Survival TNFα PIM1 JAK2 STAT3/5 P P P P P P P P P P P P TPO G-CSF JAK2 V617F JAK2 V617F Rux STAT3/5 P P P P PI3K AKT S6K S6 P P P P SCF FLT-3L LNK SOCS CBL P P P P NF  B Proliferation/Survival TNFα, GM-CSF IkBα I  B degradation IKKε IKKγ P P P P P P IBαIBα NF  B TLRs TBK1 P P IKKα IKKβ IBαIBα P P NF  B P P P P PIM1 BAD CREB RAS RAF MEK ERK Dysregulated signaling networks in myeloproliferative neoplasms Cell cycle inhibition/Apoptosis STAT1 P P STAT3/5 JAK1 STAT1 P P P P P P P P Ifn  STAT1 P P P P P P P P P P

Spectral limitations of flow cytometry can be overcome with elemental mass cytometry >30 parameters with single cell resolution Labeled cells Metal conjugated antibodies CyTOF2 mass cytometer Mass channel readout

SPADE links related cell types in a multidimensional continuum of marker expression Bendall et al Science 2011 How can we visualize data in 30+ dimensions?

SPADE identifies relevant cell subsets including HSPC CD34 median expression: Low High HSPC

Cell cycle analysis via mass cytometry Behbehani et al, Cytometry 2012

Cell cycle analysis via mass cytometry Behbehani et al, Cytometry 2012

Malumbres M, Nature Reviews Cancer 2001 Cell cycle regulators are frequently disrupted in cancer

Malumbres M, Nature Reviews Cancer 2009 Overview of CDK inhibitors in clinical development for cancer therapy Results thus far have been somewhat disappointing – why?

Suggested reading Alberts et al., Molecular Biology of the Cell, 4th Edition, Garland. Updated Chapter 17. – Malumres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer Mar;9(3): – Hanahan and Weinberg. Hallmarks of Cancer: The Next Generation. Cell Mar 4;144(5): – Anand S, Huntly BJ. Disordered signaling in myeloproliferative neoplasms. Hematol Oncol Clin North Am Oct;26(5): – Contact: