4/12/2015 The Cell Cycle Control “to divide or not to divide, that is the question”.

4/12/2015 Summary 1. The role of cell division – reproduction and growth (cell renewal and repair). 2.The mitotic cell cycle – consists of: - interphase (I=G 1 +S+G 2 ) – important steps for DNA duplication and initiation of mitosis; - mitosis (P+PM+M+A+T) – separation of two daughter cells with equal amount of genetic material (chromosomes).

4/12/2015 The Cell Cycle Control The timing and rate of cell division differ between different organisms and also between different cells of an organism. Compare skin cells with muscle or nerve cells. What is controlling the rate of cell division, how cells “know” that it is time to divide? Why cancer cells do not stop dividing?

4/12/2015 The cell cycle is regulated at the molecular level Experimental evidences suggested that the cell cycle is driven by specific chemical signals present in the cytoplasm. Most of the experiments were conducted with cell cultures. Many types of animal and plant cells can be removed from an organism and cultured in an artificial environment.

4/12/2015 Cell cycle is controlled ! Cultured mammalian cells can be induced to fuse, forming a single cell with two nuclei. The results of fusing cells at two different phases of the cell cycle suggested that particular chemicals control the progression of phases.

4/12/2015 Cell cycle is controlled ! For example, when a cell in M phase was fused with one in any other phase, the nucleus from the latter cell immediately began mitosis. If the second cell was in G1, the condensed chromosomes that appeared had single chromatids.

4/12/2015 Cell-cycle control system These experiments suggested that events happening from one cell division to another are driven by cell-cycle control system, a cyclically operating set of molecules in the cell that triggers and coordinates key events in the cell cycle When and how is the cell cycle controlled?

4/12/2015 Cell cycle is controlled ! G 1 nucleus is competent to replicate. S-phase cells contain activator G 2 nuclei aren’t competent and do not re-replicate. G 2 cells do not inhibit replication. S-phase nuclei retard mitosis in G 2 nuclei. G 2 cells do not suppress S-phase entry of G 1 -phase nuclei.

4/12/2015 Cell cycle control A checkpoint in the cell is a critical control point where stop and go signals can regulate the cycle

4/12/2015 Cell cycle control Animal cells have built-in “stop” signals that halt the cell cycle at checkpoints until overridden by “go” signals To function properly checkpoint signals have to percept “reports” from crucial cellular processes: have it been completed correctly and should the cell cycle proceed. Checkpoint also register signals from outside the cell

4/12/2015 Cell cycle control The most important decision to make is: to continue the cell division after the exit from M phase or not. Cells that do not receive the “go” signal at the G1 checkpoint, switch into a nondividing state called the G0 phase. A good example of quiescent cells are liver cells. They can be called back to the cell cycle by growth factors released during injury.

4/12/2015 Cyclins and cyclin-dependent kinases (Cdks) Regulatory molecules of the cycle transition are proteins of two main types: protein kinases and cyclins. Protein kinases are proteins that regulate the activity of the others by phosphorylating them.

4/12/2015 Cyclins and cyclin-dependent kinases (Cdks) “Go” signal at the G1 and G2 checkpoints is regulated by particular protein kinases. To be active, such a kinase must be attached to a cyclin, a protein that gets its name from its cyclically fluctuating concentration in the cell This kinases are called cyclin-dependent kinases – Cdks.

4/12/2015 Control at the G2 checkpoint The stepwise processes of the cell cycle are timed by rhythmic fluctuations in the activity of protein kinases.

4/12/2015 Control at the G2 checkpoint Cdk-cyclin complex called MPF (maturation promotion factor), acts at the G2 checkpoint to trigger mitosis. (a) The graph shows how MPF activity fluctuates with the level of cyclin in the cell.

4/12/2015 Control at the G2 checkpoint The cyclin level rises throughout interphase (G1, S, and G2 phases), then falls abruptly during mitosis (M phase). The Cdk itself is present at a constant level.

4/12/2015 Control at the G2 checkpoint (b) 1.By the G2 checkpoint (red bar), enough cyclin is available to produce many molecules of MPF. 2.MPF promotes mitosis by phosphorylating various proteins, including other enzymes.

4/12/2015 Control at the G2 checkpoint 3. One effect of MPF is the initiation of a sequence of events leading to the breakdown of its own cyclin.

4/12/2015 Control at the G2 checkpoint 4. The Cdk component of MPF is recycled. Its kinase activity will be restored by association with new cyclin that accumulates during interphase.

4/12/2015 Internal regulation Internal signals: messages from kinetochores. Anaphase, the separation of sister chromatids, does not begin until all the chromosomes are properly attached to the spindle at the metaphase plate. Certain associated proteins trigger a signalling pathway that keeps an anaphase promoting complex (APC) in an inactive state. M-phase checkpoint is the gatekeeper. Only when all the kinetochores are attached to the spindle does the “wait” signal cease.

4/12/2015 External regulation External signals: growth factors. Most of mammalian cells divide in culture only if the growth medium includes specific growth factors. PDGF – platelet-derived growth factor – is required for the division of fibroblasts.

4/12/2015 External regulation Density-dependent inhibition of cell division, a phenomenon in which crowded cells stop dividing. Cultured cells normally divide until they form a single layer of cells on the inner surface of the culture container.

4/12/2015 External regulation Anchorage dependence: to divide, cells must be attached to a substratum (extracellular matrix of a tissue). Anchorage is signalled to the cell-cycle control system via plasma membrane proteins and elements of the cytoskeleton linked to them.

4/12/2015 Cell growth is controlled cell surface growth receptors growth factor

4/12/2015 Cell cycle control 1. DNA pre-replication complexes assemble at origins 2. Inactivates APC 3. Activates transcription of S-phase CdkC components 4. Phosphorylates S- phase CdkC inhibitor Restriction points

4/12/2015 Cancer cells Cancer cells are living their own lives, they do not respond normally to the body’s control. They divide excessively and invade other tissues. When a single cell in a tissue undergoes transformation, immune system normally recognizes a transformed cell as an insurgent and destroys it.

4/12/2015 Density-dependent inhibition

4/12/2015 Cancer cells If the cell evades destruction, it may proliferate to form a tumor, a mass of abnormal cells. Benign tumor is localised at original site, malignant tumor becomes invasive. The spread of cancer cells beyond their original site is called metastasis.

4/12/2015 The growth and metastasis of a malignant breast tumor The cells of malignant (cancerous) tumors grow in an uncontrolled way and can spread to neighboring tissues and, via the circulatory system, to other parts of the body. The spread of cancer cells beyond their original site is called metastasis.

4/12/2015 Summary Cell cycle regulation – everything is under the control of Cdk: association of the kinases with cyclines support unidirectional cell cycle progression with the main influences at G1, G2 and M phases. Reading Ch 12. pp

4/12/2015 Following steps are required for p34 cdc2 (mitotic kinase) activation Phosphorylation of T-161 Dephosphorylation of T-14 and Y-15 Association with cyclin B Release of the block by CKI

4/12/2015 Pathways which determine Cdk activity Phosphorylation of Cdk itself Association with a specific cyclin Association with CKIs Association with other proteins related to cell cycle