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The cell cycle. The cell cycle is divided into four phases, G1, S, G2, and M phase. Cells that have exited the cell cycle are in a state known as G0 (zero) or quiescence, and they re-enter the cycle in early G1 phase. Cells should only divide if they have enough room, if there are sufficient growth factors present, and if they haven’t suffered undue stress or DNA damage. If conditions are appropriate, then cells make preparations for DNA replication. We don't yet understand how all of the critical information about intracellular and extracellular conditions is integrated into the decision to move forward in the cell cycle. We also don’t understand the process of cell cycle exit itself very well at all. New technologies are creating opportunities to address these gaps in our knowledge.
Once replication has started, there’s no turning back – the cell is committed to finishing the cycle. For this reason, the preparation process and the transitions from quiescence into G1 and from G1 to S phase are highly regulated and subject to a variety of checkpoint controls. Considerable evidence indicates that these regulations are corrupted in cancer cells or degenerative disorders, although the we only partly understand exactly how they are altered. To define what alterations are important, we must first understand how the regulatory mechanisms are normally supposed to work and what’s different in diseases - that's the overall goal of our research.
How do cells establish and maintain cell cycle arrest? Many cells are entering and exiting the cell cycle to maintain healthy tissues. New cells are needed for normal development and throughout our lives to replace old or damaged cells. However, cancers create new cells in places or times that they aren't needed. Normal cells remain arrested until new cells are needed, but we don't know exactly how arrest is initially established or maintained. Thus far, the field has focused primarily on how S phase genes are repressed in arrested cells. We are investigating new mechanisms that control the key enzymes and protein factors through regulated protein stability, protein interactions, and cellular metabolism.
DNA replication - balancing efficiency with tight control. To duplicate large eukaryotic genomes, DNA replication initiates at many thousands of individual locations, called origins of DNA replication. Cells must keep track of which origins have initiated (“fired”) already and which ones have not to avoid over-copying or "re-replication", a form of genetic instability that can promote tumorigenesis. Individual origins are made competent for replication by the loading of a protein complex, called MCM, during G1 phase. Origins that have MCM loaded are said to be ‘licensed’ for replication.
Once cells start S phase, MCM complexes become active DNA helicases to unwind DNA for copying. They are activated through the activity of protein kinases such as Cyclin/CDK2. At the same time all of the proteins involved in MCM loading - ORC, CDC6, and CDT1 - are then inactivated to avoid re-licensing and re-replication. This inactivation is what tightly restricts licensing to G1 phase only. These same CDK enzymes drive many steps in the cell cycle, and they are inhibited in quiescent cells by physiological signals and in cells treated with some cancer drugs.
It's worth noting that the length of G1 phase is quite variable among different cell types and different situations. Given that variability, how do cells "know" they are ready for S phase? Under what circumstances might cells be more or less ready, and what happens when they start S phase too soon? We're exploring the relationship between the length of G1 phase and how much licensing is achieved per cell before S phase starts. We're building correlations between how fast and how much MCM is loaded in cells with short or long G1 phases, and we are delving into the mechanisms that explain those correlations. Cells that have exited the cycle to a quiescent state do not license origins at all, and the mechanisms that block origin licensing during quiescence are also incompletely understood.
Single cell assays to probe dynamics. Exploring the dynamics of protein activation/inactivation and degradation/accumulation at the transitions into and out of S phase is a research area we are actively exploring. We are particularly interested in how these transitions are altered during normal cell cycle changes and in response to either extracellular signals or to the mutations associated with cancers. To deeply understand those transitions requires new experimental tools that can precisely quantify the molecular events in individual cells. Two approaches we've developed recently are analytical flow cytometry for DNA-bound proteins and live cell imaging using multiple fluorescent biosensors. These tools are revealing surprising new differences among individual cells and subpopulations of cells that were invisible by previous methods.
Flow cytometry plot of normal proliferating epithelial cells. The y axis is the amount of MCM per individual cell and the x axis is DNA content per cell. Cells are color coded blue if they have detectable MCM and orange if they have MCM and are also actively replicating DNA (S phase); grey are negative for MCM.
Progression through G1 and S phase represented as a roller-coaster track. Major decision points in G1 and at the G1/S transition are indicated in yellow, and a cell cycle checkpoint at or just prior to the G1/S transition is marked in green.
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