Cancer and the Cell Cycle

Cancer and the Cell Cycle

Outline of the lecture

What is cancer? Review of the cell cycle and regulation of cell growth Which types of genes when mutated can cancer? Roles for screening for mutations in specific genes Tumor suppressor p53 Have you figured it out?

What is cancer?

Cancer = uncontrolled proliferation of cells within the body tumor.

Tumor = clone of cells resulting from series of sequential genetic mutations loss of growth control.

Cancer is also known as malignancy. Development of cancer = oncogenesis Study or treatment of cancer = oncology

Cancer is a multi-step process.

How does this happen?

In the following slides:

= a non- dividing cell

1, 2, 3 = successive mutations, each contributing in some way to an increased rate of cell division or decreased rate of cell death.

1 Non-dividing cells

1

1 2

Non-dividing cells

2

1 1

1

1 12 2

23

Non-dividing cells

This process continues, with each successive mutation leading to a faster rate of cell division, slower rate of cell death, and eventually loss of cell adhesion.

123

1 1 1 2

1 21 2

1 2

1 123

123

123123123

123123123

Non-dividing cells

Review of the Eukaryotic Cell Cycle

The cell cycle• The cell cycle has four phases: M, during

which the cell divides; G1, during which the cell grows larger; S , during which DNA synthesis occurs; and G2, during which the cell continues to grow and prepare for mitosis. The cycle is regulated at several points.

• The restrictive point in late G1 phase is a time when the decision is made whether to continue the cycle or to to exit the cycle in a nondividing state called G0.

• Cells in G0 may differentiate and assume specialized functions.

• A cell can remain in G0 indefinitely, or it may re-enter the cell cycle in response to signals from a variety of growth factors.

The cell cycle• Once the cell passes the restriction point in

G1, the cycle will continue until it is arrested at one of several checkpoints in response to some problem that needs to be corrected.

• Progression is halted in late G1 and late G2 if DNA damage has occurred. The checkpoints allow time for the damaged DNA to be repaired before the cycle resumes.

• The checkpoint in G2 also responds to the presence of unreplicated DNA and prevents mitosis from occurring until all of the DNA has been copied.

• A checkpoint in late M phase halts the cell cycle until all of the chromosomes are properly aligned.

The Cell Cycle• Proteins from several different families

interact to regulate progression through the cell cycle.

• Cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors (CKIs) all interact either to block or unblock phases of the cycle.

• Cyclins, and Cdks act together as a dimer, functioning as the regulatory and catalytic subunits, respectively.

• Cyclins are degraded at the end of their functional period, thus inactivating their Cdk partner in the dimer.

• The assembly of the dimers is regulated by other proteins.

The cell cycle• In the event that a cell enters an S

phase with damaged DNA, apoptosis may be triggered to prevent the mutant cell from reproducing itself.

Seven levels of regulation of cell growth

An unrepaired mutation in a gene for a DNA-repair protein, a cell-cycle control protein, or an anti-apoptosis protein can increase the likelihood of a cancer developing.

An Example of Cell Cycle Regulation by a Serum Growth Factor Cyclin D is made following the

binding of the serum growth factor to its receptor and the ensuing cascade of phosphorylations.

An Example of Cell Cycle Regulation by a Serum Growth Factor

An Example of Cell Cycle Regulation by a Serum Growth Factor

Cyclin D associates with either Cdk4 or Cdk6.

P16 may block the assembly. After assembly the Cdk becomes

phosphorylated. This may be blocked by either p21 or p27 The target of the active dimer is Rb which is

bound to a transcription factor called E2F. The Rb/E2F dimer blocks transcription of

genes needed to enter the S phase. Phosphorylation of Rb results in its

dissociation from E2F.

An Example of Cell Cycle Regulation by a Serum Growth Factor

This results in activation of S phase genes.

In addition to its ability to block the association of cyclin D with a Cdk, P16 can also directly block the phosphorylation of Rb.

Phosphorylation of Rb

An Example of Cell Cycle Regulation by a Serum Growth Factor

P16, p21, and p27 are regulated by p53 (more on this later) which blocks the cell cycle in the G1 phase if there is DNA damage.

P53, Rb, p21, p16, and p27 are called tumor supressors because their normal function is to prevent the growth of cells with damaged DNA.

An Example of Cell Cycle Regulation by a Serum Growth Factor

P53 also responds to unrepaired DNA damage by triggering apoptosis of the injured cell.

It interacts with a member of the Bcl-2 family of proteins which, in turn activate special enzymes called caspases.

Caspases initiate a protease cascade that results in digestion of the DNA.

This ultimately leads to cell death.

Apoptosis

Which types of genes when mutated can cancer?

Oncogenes = genes whose products turn DNA synthesis ON

Tumor suppressors/anti-oncogenes = genes whose products turn DNA synthesis OFF

Genes whose products contribute to genomic stability

Genes whose products contribute to cell longevity

Which types of genes when mutated can cancer? Oncogenes (turn DNA synthesis ON)

In progression towards cancer, a gene for a protein that normally stimulates DNA synthesis (proto-oncogene) is either consitutively expressed at high levels or mutated such that protein product is constitutively

active, i.e., can not be inactivated Classes I-IV from Slide # 15 generally give rise to

dominantly active oncogenes. Examples: see next slide.

Oncogenes

Which types of genes when mutated can cancer? Tumor suppressors/anti-oncogenes (turn DNA

synthesis OFF) In the progression towards cancer, a gene for a

protein that normally inhibits DNA synthesis is either permanently inactivated or mutated such that the protein product is inactive

Mutations in Class VI, cell-cycle control proteins, from Slide #15.

Examples: APC inhibits Wnt gene product from activating myc Rb inhibits activation of transcription of DNA synthesis

genes

Which types of genes when mutated can cancer?

Contributors to genomic stability Some tumor suppressors turn DNA synthesis off when DNA is damaged. The progression toward cancer occurs when a gene for a protein which

contributes to DNA repair is permanently inactivated or mutated such that protein product is inactive

Mutations in repair genes increase likelihood of mutations in proto-oncogenes and tumor suppressors.

Examples: p53 gene product induces genes for DNA repair MDM2 gene product destabilizes p53 MutS and MutL gene products repair UV or chemically damaged DNA

Which types of genes when mutated can cancer?

Contributors to cell longevity (anti-apoptosis genes) Progression toward cancer can occur when an anti-

apoptosis gene is constitutively expressed or mutated such that protein product is constitutively

active Allows survival of cells with oncogenic mutations Example: Bcl2

Roles for screening for mutations in specific genes

To determine Type of cancer Familial predispositions Progression of the cancer

What is p53? A protein of ~53 kilodaltons A nuclear phosphoprotein

What is p53? Transcriptional regulator

Binds to 12 bp recognition sequence in the promoters (regulatory regions) of the genes it regulates

Activates transcription by interacting with RNA polymerase complex

What is p53? Acts as a tetramer

Individual molecules associate at tetramerization region

Oligomerization of mutated p53 with wt p53 inactive p53 complex

What is p53? Binding of damaged DNA fragments to p53

causes p53 to be stabilized and accumulate in the cell.

When damaged DNA is not present, p53 is turned over rapidly and does not accumulate because the protein MDM2 binds to the transcription-

activation region of p53 and targets p53 for degradation by a proteosome.

Note: MDM2 binds when the TAD is NOT phosphorylated.

(TAD)

p53 as a transcriptional regulator If DNA damage is detected by binding of DNA fragments to the

non-specific DNA binding region of p53, p53 stops DNA synthesis until the damage is repaired.

If DNA damage is detected, then p53 is phosphorylated by a protein known as ATM MDM2 is released from being bound to the transcriptional activation

domain of p53 and p53 is able to act as a transcriptional activator and turn on genes for

cyclin dependent kinase inhibitor p21, which• stops or prevents DNA synthesis

DNA repair • Example: GADD45

If DNA damage is extensive and can not be repaired, p53 induces genes for apoptosis (programmed cell death).

p53 as a transcriptional regulator p53 activates the gene for MDM2

MDM2 targets p53 for degradation and prevents inappropriate

build up prevents transcriptional activation by p53

So, it’s a negative feedback loop!

p53 also turns expression of some genes off.

How does p53 inhibit DNA synthesis? Let’s work backwards.

E2F transcription factor turns on transcription of genes for DNA synthesis.

E2F can’t turn on genes if it is bound to Rb, a tumor suppressor.

Rb can’t bind E2F if it is heavily phosphorylated.

Rb is phosphorylated by cylin-dependent kinases (CDKs).

How does p53 inhibit DNA synthesis? Cylin dependent kinases can be inhibited by cyclin

dependent kinase inhibitors (CDKIs). If CDKs are inhibited Rb won’t be phosphorylated E2F will be bound by Rb DNA synthesis gene will not be transcribed

And remember . . . . P53 induces expression of CDKI p21, a cyclin

dependent kinase inhibitor! Check out the next slide for a visual of these

pathways.

Phosphorylation of Rb

Figure legend on next slide.

p53 Mutations - where are they?

This magnification of mutations in the DNA binding region of p53 gives more information regarding how the mutation affects p53. Note particularly that some mutations cause p53 to be misfolded (denatured) and others do not.

Have you figured it out?For our assay, the samples are cell extracts from two mouse

cell lines, BC3H1 and C2C12.

One line is wild type for p53; one is mutant.One accumulates detectable levels of p53; one

doesn’t.

Based on this lecture and your assay results, have you figured out which cell line does what?