Mammalian cell cycle

In order to sustain a tissue its cells need to fulfil three fundamental functions. Those are proliferartion, differentiation and cell death. The cell cycle is the highly coordinated sequence of those events that happen during an in between one cell division. Cell cycle itself consists two general phases. The shorter M-phase (or mitosis) is when the duplication of the chromosomes and the actual cell division take place. The Interphase is almost 10 times longer than mitosis and defined through cell growth and various

metabolic activities (140). Both interphase and mitosis are further subdivided into synthesis enzymes (DNA-polymerases, ligases etc.). During S-phase the cell duplicates its genome and after this process each chromosome consists once again of two sister chromatids. The last phase of the interphase is the so-called postsynthetic or premitotic G₂-Phase. Here the cell is preparing for the upcoming cell division by retracting extracellular contacts and taking up liquid in order to gain more volume. After completing the three stages of the interphase the cell is now ready to divide once again and enters mitosis. Mitosis itself is very short and only takes around 20 minutes. It is subdivided into 5 highly orchestrated phases named pro-, prometa-, meta-, ana-, and telophase. Upon completion of mitosis the cell either continues with the next G1-phase or exits the cell cycle to enter the so-called G₀-phase. The G₀-phase is a resting phase in which cells do not progress further through the cell cycle but remain static. This state can either be permanent or reversible depending on the cell type and differentiation state. Cells in G₀ that are able to re-enter cell cycle are called “quiescent”. Those cells (e.g. hepatocytes) are able to proliferate again upon certain stimuli even after weeks and months in G0. Other cells (e.g. neurons) are not able to re-enter cell cycle once they are fully differentiated and thus are called “postmitotic”.

2.7.1 Cell cycle regulation and checkpoints

The cell cycle is an immensely coordinated biological process. To deal with such a complicated task in an orderly fashion an intricate ensemble of cell cycle–associated proteins is used. Those include cyclins, cyclin-dependent kinases (CDKs), kinases and phosphatases which all work together either in synchrony or rapid succession to ensure cell cycle progression. Cyclins and their respective kinases form complexes to exert their function. Each cyclin is expressed in a specific period during cell cycle and initiates the transition to a new cell cycle phase (142). After that it is rapidly degraded again while other cyclins are upregulated. If certain mitosis inducing factors (mitogens) are present in G1-phase the D-cyclins (CCND1, 2, 3) form a complex together with their specific protein kinases CDK2, 4 and 6 which is called SPF (S-phase promoting factor). For the transition from late G1- to S-phase another complex (cyclin-E-CDK2-complex) is needed. Shortly before the start of DNA replication cyclin A (CCNA) is upregulated. CCNA also activates CDK2 and both build a stable complex until the end of G₂-phase. During late S- and G₂-phase cyclin B (CCNB) is upregulated and associates with CDK1 to form the MPF (mitosis promoting factor). However, the phosphorylation of CDK1 keeps this complex inactive until the end of G₂-phase. Upon dephosphorylation through the protein phosphatase CDC25 the CDK1-CCNB-complex is translocated into the nucleus where mitosis is initialized. There, the complex stays active until CCNB is degraded during meta- to anaphase transition.

Figure 6: cell cycle-dependent activity of CDK-complexes (adapted from Müller-Esterl, 2004)

To ensure a functional cell cycle there is also a vast amount of negative cell cycle regulators. They can be roughly divided into two groups. The first group is comprised of the CDK inhibitory proteins (CIP). By inhibiting all cyclin-kinase-complexes, they stop G_0/1- to S-phase transition. One example for a CIP-family member is the p27 protein. P27 controls the transition from G0- to G1-phase. It is expressed in postmitotic cells and prevents cell cycle re-entry. The second group of inhibitors is called Ink4 (inhibitor of kinase 4). They solely inhibit the CDK4/6-CCND-complexes and stall G1- phase progression. Another type of cell cycle regulation is provided by tumour suppressor genes like the retinoblastoma protein (pRb). pRb also controls the transition from G₁- to S-phase and therefore prevents the replication of damaged DNA. In its unphosphorylated state pRb encapsulates the transcription factor E2F which in turn is essential for the induction of the S-phase. Only upon phosphorylation by CDK4/6-CCND-complexes during G1-phase does pRb release E2F. The transcription factor then binds to the promoters of genes that are important for DNA replication (like DNA-polymerase) and activates their transcription.

Cyclins, CDKs and cell cycle inhibitors provide the basic schedule for normal cell cycle regulation. However, certain external and internal factors are able to stop cell cycle progression at specifically implemented checkpoints. Those are necessary to control for incomplete or erroneous cell cycle phases. External factors that can lead to cell cycle arrest and G₀-phase entry are for example cell culture density or insufficient supply of nutrients. Internal factors like critical cell mass, DNA damage and incomplete replication can also induce a proliferation stop or even apoptosis. Each of these factors triggers one or more checkpoints at specific stages during cell cycle. DNA damage events for example activate cell cycle checkpoints before (G₁-phase), during (mid-S-phase) and after (G2-phase) replication. Another critical step is the so-called

“spindle checkpoint”. It monitors the alignment of the chromosomes on the equatorial plate and the connection of spindle fibres to the kinetochore during metaphase. In the subsequent anaphase, the two sister chromatids of the chromosome are separated; but only if all chromosomes are correctly aligned and the spindle fibres are connected to both centromeres via the kinetochore.

Those checkpoints are the “safeguards” against improper cell division and DNA replication which might lead to uncontrolled cell proliferation and cancer. But what if

they detect irregularities in the cell cycle program and repair mechanisms fail to correct the mistake? In this case the cell goes into a self-destruction mode also known as apoptosis.

2.7.2 Cell death

Apoptosis is a tightly and actively controlled process that is crucial for many physiological functions like limb development. This makes it fundamentally different from necrosis, an involuntary and chaotic cell death often caused by severe external damage of tissue (e.g. sunburn). The process of apoptosis itself can be grouped into three different phases; initiation phase, effector phase and execution phase. Although there are many different stimuli that are able to trigger apoptosis, there are only two well-studied signalling pathways that lead to the induction of the process; the intrinsic and the extrinsic pathway. The extrinsic pathway is executed by the so-called “death – receptors” of the tumour necrosis factor (TNF) superfamily. The natural ligands of TNF receptors are TNF itself and other cytokines. TNF-receptors do not possess an enzymatic activity. That is why they recruit cytoplasmic adaptor proteins or “death domains”. They form the DISC (death inducing signalling complex) which starts a proteolytic signalling cascade that leads to cell death. The intrinsic pathway can take place with or without the help of death receptors. Internal signals like DNA damage or oxidative stress lead to caspase activation (see below) which in turn activates proteins that influence mitochondrial permeability. The increased permeability of the outer mitochondrial membrane leads to the release of cytochrome c into the cytoplasm. This activates further caspases and finally initiates apoptosis. In both extrinsic and intrinsic pathway, caspases play a decisive role. Caspases are intracellular protease enzymes that cut their substrate proteins selectively at aspartate residues. To date there are 14 different caspases known in man, with 7 of them involved in apoptosis. Caspases are expressed as inactive proenzymes which are activated through auto-proteolysis or other proteins. Some caspases participate in the induction of apoptosis (initiator caspases).

Those include the caspases 2, 8, 9 and 10. Others operate downstream of an amplifying proteolytic signal cascade and are involved in the final apoptotic degradation process of the cell. They are called effector caspases and include the caspases 3, 6 and 7. Apart from caspases there is a whole plethora of apoptotic and antiapoptotic molecules that help to regulate the apoptotic sensitivity and the apoptotic program of a cell (143).

2.7.3 Cell cycle, apoptosis and disease

If apoptotic and/or cell cycle regulation goes wrong, it can lead to severe problems in cellular function. On the opposing ends of this large spectrum of cellular defects are cancer or degeneration diseases (144). Therefore, controlling the balance between degeneration and proliferation helps to maintain a functional equilibrium in the body.

As mentioned above postmitotic cells have exited the cell cycle and are no longer able to re-enter it upon stimulation. Therefore they should not be affected by cell cycle deregulation and subsequent proliferative signals. It was shown however that cell cycle induction in postmitotic neurons leads to cell death (145). This finding is already more than 20 years old and since then researchers found evidence for cell cycle-related events in Alzheimer`s disease (AD), Parkinson`s disease (PD), amyotrophic lateral

sclerosis (ALS) and stroke ataxia-telangiectasia (AT) (146-148). Based on this, some hypothesized that the cell cycle induction seen in many neurodegenerative diseases is due to a deregulation of apoptosis and/or cell cycle; a process often seen in cancer phenotypes of other tissues (149). What strengthens this theory is that to date no cancer of real postmitotic origin has been reported. Re-entrance into cell cycle is associated with neuronal cell death in a number of mouse models (150,151) and in vitro studies (152,153). Apart from models that are not disease-related, several transgenic AD mouse models also show abnormal cell cycle processes including expression of cell cycle proteins and DNA replication (154). Recently, it was shown that by blocking cell cycle, cell death could be averted in cultures of neurodegenerative disease models (155).

But despite the availability of models that simulate a cell cycle-related neurodegeneration, it is still unclear what ultimately causes cell death in the real disease pathology. Some propose cell death as consequence of genomic instability (aneuploidy) caused by DNA replication (156). This would explain the high incidence of AD pathology in Down syndrome patients (157). Another possible explanation could be provided by the observation that cell cycle deregulation in postmitotic cells has a synergistic effect together with other cellular insults. Neurons treated with UV to induce DNA damage also need irregular cyclin activity to introduce cell death (158).

This observation led to the so-called double-hit hypothesis. In short, an irregularity like heightened oxidative stress levels or elevated cell cycle markers can persist for years without the cell going into apoptosis. Only after the second insult of the two takes place does apoptosis occur. This accumulative effect might explain the late onset of those neurodegenerative disease types.