Cell cycle – Core mechanism CDK+Cyclin APC & SCF E3 UB ligases

Modulation of the basic cell cycle control system Checkpoint control system – cell cycle is controlled by STOP & GO – process is assessed & possibly halted – signals indicate if key cellular processes have been completed correctly

Summary of CDK regulation mechanisms

Checkpoint control is achieved by CDK regulation

How DNA damage arrests the cell cycle? E3 Ub ligase

What starts the cell cycle?

Petri plate Scalpels Without serum With Serum Cell/ Tissue culture What starts the cell cycle? Collagenase/ protease Cells need soluble factors from serum to proliferate and survive

Adherence and contact inhibitioncontact inhibition Density-dependent inhibition, in which crowded cells stop dividing What starts the cell cycle?

LE 12-18a Cells anchor to dish surface and divide (anchorage dependence). When cells have formed a complete single layer, they stop dividing (density-dependent inhibition). If some cells are scraped away, the remaining cells divide to fill the gap and then stop (density-dependent inhibition). 25 µm Normal mammalian cells

Most animal cells also exhibit anchorage dependence, in which they must be attached to a substratum in order to divide

Cancer cells do not exhibit anchorage dependence or density-dependent inhibition. Cancer cells 25 µm

Quiescent cells are found in G0

Extracellular control of cell proliferation Cells communicate with their environment to decide when to proliferate. Major signals to cells include: Survival factors - inhibit apoptosis Growth factors – promote biogenesis/growth Mitogens - activate cell cycle

Survival factors - inhibit apoptosis

Growth factors – promote biogenesis

Mitogens - activate cell cycle

D-type cyclins are highly regulated by external signals Cyclin D RNA after serum stimulation of starved cells:

Mitogens - activate cell cycle

D-type cyclins are highly regulated by external signals

Decision about G0/G1 is made during early G1

Figure 8.12 The Biology of Cancer (© Garland Science 2007) Restriction point After CDK/CycD activation and R the cell cycle program becomes cell autonomous and does not respond to external signals

Activation of CycD-CDK leads to phosphorylation and inactivation of RB In resting state RB bind and represses the transcription factors from the E2F family

Activation of CycD-CDK leads to phosphorylation and inactivation of RB Rb phosphorylation releases E2Fs and allows transcription

Two phosphorylation stages in RB activation

The CDK-Cyclin complex activates events of the cell cycle G1= CDK inactive, APC/C active, Rb active and blocks E2Fs

Figure 8.12 The Biology of Cancer (© Garland Science 2007) Once G1/S CDK (E-CDK2) is active the positive feedback drives the process forward until completion and return to G1 (APC/C active, Rb active and blocks E2Fs)

G1: APC-Hct1(Cdh1) active CDKi present and active Rb is active and inhibits activity of E2F TFs Transcription of G1/S,S cyclins is repressed Activation of G1-CDK causes Rb phosphorylation and activation of E2Fs Positive feedback loop enhances E2F activity: E2F activate E2F transcription G1/S CDK enhances phosphorylation of Rb, and CDKis Start of Cell cycle

Feedback loops in the G1-S transition E2F activates CycE, A and CDK2 - positive FB CycE/CDK2 inactivates p27 - positive FB CycA/CDK2 inactivates E2F- Posphorylates CycE negative FB SCF

Although the cycle can not be reversed, it can be halted at checkpoints E3 Ub ligase

E2F transcription factor family: repressors and activators repressors activators E2F7,8 Trimarchi and Lees, 2002 NR MCB 3:11

E2F transcription factors activate many S-phase associated genes From Knudsen NRC 2008

Retinoblastoma - the first genetically dissected familial cancer Retinoblastoma is a rare pediatric tumor arising in precursors of photoreceptor cells in the retina:

Rb inhibits E2F function Blocks recruitment of transcription activators

Figure 8.35 The Biology of Cancer (© Garland Science 2007)

Replication 1.How is the genome replicated ? 2.What are origins and how are they assembled ? 3.How are initiation and elongation regulated ? 4.How is re-replication prevented ?

The human genome is encoded by 3,000,000,000 bp (3x10 9 ) which must all be replicated within a few hours with the highest fidelity. The loss of a bp is irreversible and a single mistake could potentially lead to the death of the organism.

Analogy One page contains about 3000 letters. Imagine that you have to copy one million pages in a few hours and have to bind them into 23 volumes. You can use as many typists as you like but you are allowed no mistakes -no missing letter, no letter twice, and no substitutions.

Each eukaryotic chromosome is one linear DNA double helix Average ~10 8 base pairs long DNA replication initiates at many different sites simultaneously.

Prevention of re-replication S-CDKs can not promote replication in G2 cells Replicated Chromosomes are different from unreplicated chromosome Replication licensing

Figure Molecular Biology of the Cell (© Garland Science 2008)

Figure Molecular Biology of the Cell (© Garland Science 2008)

Only origins loaded with MCM are licensed to replicate ORC+Cdc6 =clamp loader

Once replication initiates MCM is displaced from the origin and this origin losses license MCM complex functions as a helicase

Mechanisms to prevent re-replication: S-CDK Pre-replication complexPre-initiation complex

Separating pre-replication from pre-initiating ensures correct replication

Once replication initiates MCM is displaced from the origin and this origin losses license MCM complex moves along the DNA ahead of the fork and are essential for replication All CDKs can block replication, defects in CDK activity allow re-replication

Figure (part 3 of 3) Molecular Biology of the Cell (© Garland Science 2008)

In mammals: Geminin participates in prevention

Geminin Is a substrate for APC/C Immediately after anaphase, APC/C degrades Geminin and Cyclins and licensing is restored The Pre-replication complex is reorganized