Cancer and Oncogenes

Cancer is a diverse group of diseases with one common characteristic: unchecked cellular replication.  Via several potential mechanisms, cancer cells are able to avoid all of the checkpoints involved in cell growth and division, thus enabling them to divide more frequently or indefinitely.  Many events can lead to the development of a cancer cell, including inheritance of mutated DNA or the activity of a carcinogen, or a chemical agent that leads to the development of cancer.

Gene expression is often deregulated in cancer cells such that some genes are overexpressed, while others are underexpressed.  Genes that can be mutated to lead to an upregulation of activity and lead to the development of a cancer cell are termed proto-oncogenes.  When these proto-oncogenes are actually mutated, they are considered oncogenes.

An oncogene is often a gene involved in regulating cell division and drive the cell cycle.  When they are overexpressed, such as during cancer, they can push the cell to divide more frequently and, with further mutations, transform the cell such that it divides without restriction.

Oncogenes were first discovered in viruses, specifically in Rous Sarcoma Virus (RSV), a retrovirus that encodes a homologue to cellular src kinase (the viral form called v-src).  Tumors in birds caused by RSV are the result of v-src causing unregulated cellular proliferation.  Large amounts of research into this area has identified cellular src kinase as a proto-oncogene that, when mutated to become constitutively active, becomes an oncogene and can drive cancer development.  Interestingly, viruses have highlighted a number of cellular oncogenes and pathways that are improperly regulated in cancer.  Over 20 viral oncogenes have been identified to date.

Cellular proto-oncogenes (the genes before they become oncogenes) can promote cellular proliferation and the development of cancer in several ways.  One of these ways is to be overexpressed and function when the gene product really shouldn't function.  This is the case with proteins such as myc and growth factor receptors.  With overexpression of these proteins, there is the potential for amplified signaling through these pathways that can push the cell to divide more than it normally does, leading to the development of cancer.  An additional mechanism whereby a proto-oncogene can become an oncogene is via mutation that leads to improper regulation, such as constitutive activity.  A classical example of this type of phenomenon is via Ras, which when mutated is constitutively active and cannot hydrolyze an attached GTP to inactivate.  Thus, Ras remains active and cannot be "turned off."  This constant activity of Ras results in  signal transduction to the nucleus of the cell and pushes the cell to divide through transcription of several genes involved in cell division.

Cell Cycle III: Entering and Exiting Mitosis (and the Events Between)

Today is the final part of the cell cycle that I will be writing, though more information about cell division will be posted eventually in the meiosis study notes.  I find that the pathways involved in mitosis are rather easy to understand, as everything fits together - it just takes some effort to realize how everything works with everything else. 

When the cell is ready to begin anaphase, the sister chromatids must be aligned at the metaphase plate, and the proper Cdk/cyclin must remain activated (Cdc2/cyclin B).  When anaphase is to begin, APC/C (anaphase promoting complex / cyclosome) activates via phosphorylation from Cdk/cyclin and association with a cofactor.  APC/C acts as a ubiquitin ligase and is involved in poly-ubiquitinating proteins to target them for degradation.  When phosphorylated APC/C binds Cdc20, it targets securin for degradation.  Securin typically holds a protein called separase.  When securin releases separase, the protein cleaves cohesion complexes and separates sister chromatids.  This prompts the cell to enter anaphase when the sister chromatids migrate away from each other.  This process is regulated by Bub and Mad2, which act to inhibit the action of APC/C^Cdc20.  When a checkpoint is activated by a sister chromatid not being bound to a kinetochore MT, for example, it actively signals to Bub, which activates Mad2 to activate Mad2*.  Mad2* is involved in inhibiting the APC/C^Cdc20 complex.  The end result is that the sister chromatids do not separate. 

During anaphase, the microtubules are undergoing a number of changes.  Initially in anaphase (Anaphase A), the kinetochore microtubules, bound to the kinetochores of the sister chromatids, begin to shorten, pulling the chromatids apart.  During anaphase B, the astral microtubules remain in place, maintaining the spindle pole in place.  The polar microtubules begin to push on each other by elongating, which pushes the spindle poles apart and promotes separation of the nuclei.  When the spindles have moved apart and the chromatids have separated, APC/C targets Ase1p for degradation, which leads to breakdown of the spindle. 

When cohesion is cleaved by separase (see above), Cdc14 phosphatase is released from the nucleolus.  This event is the signal to end mitosis by inducing expression expression of Sic1, which acts to deactivate MPF.  Cdc14 dephosphorylates and activates Cdh1, which complexes with APC/C to target MPF for degradation.

With MPF degraded proteolytically, the nuclear envelope is free to reform.  Constitutive phosphatases dephosphorylate the lamins and nuclear pore complexes.  This results in the formation of karyomeres, which are small vesicles (with nuclear pores in them) that form around the sister chromatids.  The karyomeres then fuse to reform the nuclear envelope. 

In addition to the nuclear lamins and pore complexes, myosin light chain is also dephosphorylated when MPF is degraded.  This results in the activation of the protein and the initiation of cytokinesis when the contractile ring begins to form.

A quick summary…

Phase of the Cell Cycle	Description
Interphase	The cell is preparing to duplicate; centrosomes appear outside the nucleus
Early Prophase	Spindle poles form and sister chromatids condense
Late Prophase	Sister chromatids are condensed and begin to be attached by kinetochore MTs
Metaphase	Sister chromatids align at the metaphase plate
Anaphase	The cell begins to divide the sister chromatids by releasing them to each pole
Telophase	The nuclear envelope reforms and the cells begin to structurally split

The cell cycle isn’t so scary, but it certainly is complex, and not all of the components and regulatory mechanisms have been discussed here (or discovered in the literature either!).  Nonetheless, it is crucial to understand the basics of the cell cycle because it is so intimately related to nearly all biological processes.

Cell Cycle II: Condensins, Cohesins, Microtubules, and All the Fun Stuff

I'm going to try to make this blog post a little different with a short introduction before jumping right into my writings and ramblings about the cell cycle.  This post is the second of three concerning the cell cycle, though more information will be eventually posted that is somewhat related to this topic (notably the meiosis notes).  I haven't drawn a diagram for this particular post, but there are not many mechanisms involved.  Tomorrow's post will include a cool drawing of separase, securin, and a bunch of other cool stuff.  

When I was in college and learning the cell cycle, my professors weren't interesting or very organized.  Therefore, I've tried to organize this in a way that makes sense to me.  Since this organization might not make sense to everyone else, I'll be including a list of links to review articles and other websites that concern the cell cycle when all of the sections have been posted.

Now that we’ve covered the “motor” of the cell cycle, we need to consider regulation in terms of different stages of the cell cycle and the associated checkpoints.  These checkpoints influence the activation of Cdk/cyclin and affect whether the cell arrests or progresses through the cycle.  During S phase, the cell must check that the centrioles are duplicated and MPF has built up.  Before it is able to leave S phase, it must recognize that all DNA has been replicated faithfully and that there has been no damage accumulation. 

One of the important requirements for S phase to proceed is for sister chromatids to be attached to each other.  This cohesion is facilitated by several proteins named cohesins that loop around both chromatids and “tie” them together.  Smc molecules contain a hinge and ATPase domain:  Smc3 and Smc1 together wrap around the sister chromatids like a cord.  Scc1 and Scc3 act as the latches on the ends of the Smc molecules.  In yeast, the cohesions exist on the chromosome until anaphase and the yeast chromosomes do not become highly condensed.  In mammalian cells, however, the chromosomes become condensed via the condensin complex.  Condensins consist of Smc2 and Smc4 loops along with CAP-G, -H, and –D2.  The condensin complex acts to supercoil DNA.  These sister chromatids, which are connected by the cohesin or condensin complexes are held in place by kinetochore microtubules.  These microtubules emanate from the centrosome and connect to the sister chromatids via the kinetochore.  Additionally, the cell contains astral microtubules that connect the centrosomes to the cell periphery.  Polar microtubules connect to each other near the metaphase plate and act to push on each other, thereby separating the two poles of the cell.

When the cell is in metaphase and the sister chromatids must all align on the metaphase plate, the kinetochore microtubules pull on the chromatids.  These chromatids then align by moving back and forth until they have reached a point where the tension is equal in both directions.  Chromatids that are not attached to kinetochore microtubules actively signal (beep) that the sister chromatids are not prepared for separation (in anaphase).

When the cell is ready to split, its DNA content is store in the nucleus, which is surrounded by the nuclear envelope, made of lamins.  MPF phosphorylates these lamins to disrupt the lamina.  When phosphorylated, the lamins vesiculates and dissociate.  Additionally, nuclear pore complexes dissociate as they, too, are phosphorylated.  This allows for the duplicated DNA to separate to the separate poles of the two daughter cells.

As mentioned previously, Cdc25 is the activating phosphatase for Cdk/cyclin.  During interphase, it is phosphorylated on serine 216 and bound to a 14-3-3 protein.  This protein blocks Cdc25’s NLS (nuclear localization sequence) and exposes its NES (nuclear export sequence), causing Cdc25 to be cytoplasmic.  During mitosis, the phosphorylation of Cdc25 changes and 14-3-3 no longer blocks.  This change exposes the NES, and Cdc25 is phosphorylated by Pin1 to upregulate its activity.  This drives the activation of Cdc2/cyclin B, which is active during mitosis only.

Cell Cycle I: Cdks and cyclins, OH MY

The Cell Cycle

Understanding the cell cycle is critical to comprehending the many other processes that occur.  A myridad of events, such as DNA damage or cell-to-cell signaling, will affect the frequency of a cell’s division, and, as well, the cell cycle will also impact several pathways within the cell.

DNA replication is central to the cell cycle as well, as it must occur once only during the cell’s S phase.  However, before it can do this, the cell must receive growth signals, consider its size and nutrient availability, and determine if there is any DNA damage.  Several checkpoints have been built into the cell cycle prior to S phase to prevent the cell from unnecessarily replicating its DNA, which is a very costly process energetically.   Thus, the cell would not want to waste its resources if it cannot complete replication or if the DNA is damage.  After all, what would be the use of replicating if the cell’s DNA is damaged and progeny cells may not even survive?  Additionally, synthesis of the DNA must also be coordinated with replication machinery that will actually perform the reactions necessary.  Finally, regulators of replication initiation complexes (see notes on DNA replication for information about initiation complexes) must be phosphorylated or synthesized to prepare the DNA for S phase.

Much of the original research into the cell cycle took place in yeast, either of the budding or fission variety (S. cerevisiae or S. pombe, respectively), due to the ease of identifying mutants that were unable to progress through the cell cycle.  Due to yeast’s morphology, one is able to easily decipher which stage of the cell cycle it is in.  For example, in budding yeast, one is able to tell that the cell is in S phase when it has just begun to schmoo and M phase when its chromosomes are segregated.  As mentioned, the original studies in cell cycle considered mutants that were unable to complete the cycle and arrested at different stages.  This was performed by using temperature-sensitive (ts) mutants of the yeast.

Isolating temperature-sensitive cell cycle mutants simplified:

1.      Mutagenize with your favorite mutagen

2.      Screen for temperature sensitive mutants by replica plating

3.      Look for cells that arrest at a uniform stage of the cell cycle

4.      Sort mutants into complementation groups

5.      Transform mutants with plasmid library to identify gene of interest

Microscopically, it is relatively easy to determine when a temperature-sensitive mutant arrested in the cell cycle due to the distinct morphology.  Cells that arrest in the same phase of the cycle look the same, regardless of whether there are nutrients present or not.  For example, if a cell had arrested in metaphase in mitosis, all of the cells would appear to be schmooing with their chromosomes lined up at the metaphase plate when they are placed at the non-permissive temperature (36^o).  When grown at the permissive temperature (25^o), the cells will continue the cell cycle and will not be synchronized.

The molecular basis of cell cycle control

A great number of molecules are involved in the cell cycle and its regulation.  However, the main complex that is the driving force of the cell cycle is Cdk-cyclin. Cdk, or cyclin-dependent kinase, phosphorylates a number of targets when it becomes activated, and it is only active when it is bound to cyclin, which is a short-lived protein in the cell that is present only during cell division.  In fact, there are two types of cyclins (in yeast): S- and M-cyclin, for synthesis and mitosis cyclins.  Their different binding affinities and abundances during different stages of the cell cycle give Cdk its specificity.  Cdk-cyclin can also be called MPF, for mitosis-promoting factor, when it is the engine driving the G2-to-M transition, or SPF, for S-phase-promoting factor, when used for the G1-to-S transition. 

Cyclin binding isn’t the only requirement for Cdk to become active.  In fact, there are a number of additional steps that must occur for the enzyme to phosphorylate its targets.  Two of the first proteins identified that affects Cdk’s activity were Cdc25 and Wee1.  Wee1, when expressed in excess, led to elongated  yeast cells; while Cdc25 excess led to small cells (cells that passed through mitosis quickly).  Thus, Wee1 acts to inhibit MPF (Cdk-M-cyclin) and Cdc25 activates it.  The exact mechanisms was found to be phosphorylation and dephosphorylation:  Wee1, a kinase, adds an inhibitory phosphate to Cdk on tyrosine 15; Cdc25 phosphatase removes this inhibitory kinase and promotes progression through the cell cycle.  Cdk required both the removal of the inhibitory phosphate and the binding of cyclin to become partially active, but this, too, is not all for a fully active enzyme.  In addition, Cdk must be phosphorylated by a Cdk-activating kinase (CAK), which adds an activating phosphate group to make the enzyme fully active.  This phosphorylation event at threonine 161 on Cdk results in the extension of the T-loop to allow substrate binding and facilitate catalysis.

Due to the importance of Cdk-cyclin for the cell, it has several layers of regulation, and there are many other factors other than phosphorylation, as described above, that regulate it.  When Cdk-cyclin is associated with a CDK inhibitor (CKI) such as p27 (mammalian cells) or Sic1 (yeast), its activity is blocked.  Another CKI is p21, which is involved in DNA damage response.  When DNA damage is detected and p53 is activated, p21 is active and binds Cdk/cyclin to prevent cell cycle progression. CKIs are deactivated by SCF, which bind and ubiquitinated CKI when it is phosphorylated (CKI acts as a phosphordegron – it is targeted for degradation upon phosphorylation).  When SCF is active and ubiquitinates the CKI, Cdk-cyclin can become active.  Sic1 in yeast is involved in binding the S-phase cyclin and Cdc28 (the Cdk), and it is phosphorylated by the Cdk-cyclin present during G₁.  In this way, the Cdk-cyclin that is active prior to S-phase cyclin/Cdc28 works to activate the next stage in the cell cycle.

Review: Regulation of Cdk/cyclin

·         Cyclin must bind Cdk for activity

·         Activating phosphate (added by CAK)

·         Inactivating phosphate (added by CKI/Wee1; removed by Cdc25)

·         Inactivation by p21/27

·         p21 is stimulated by p53 during DNA damage

Cyclin D is an important cyclin molecule involved in cell cycle progression that binds Cdk4/6.  When this complex is active, the Rb protein is phosphorylated.  Normally, Rb will be found to E2F, an important transcription factor.  When Rb is phosphorylated, it dissociates from E2F, which then acts as a transcriptional activator to upregulate transcription of Cyclin E / Cdk2 as well as its own transcription and much of the replication machinery.

The cyclincs and Cdks of mammals:

Stage	Cdk	Cyclin
G1	Cdk4	Cyclin D
	Cdk6	Cyclin D
G1 – S	Cdk2	Cyclin E
S	Cdk2	Cyclin A
G2 – M	Cdc2 (Cdk1)	Cyclin B