Hanahan and Weinberg, Cell 144:646 (2011)

The Hallmarks of Cancer

Hanahan and Weinberg, Cell 144:646 (2011)

Newer Hallmarks of Cancer

Hanahan and Weinberg, Cell 144:646 (2011)

Therapeutic Targeting of the Hallmarks of Cancer

Cell Regulatory Networks Important in Cancer Cells

Hanahan and Weinberg, Cell 100:57-70 (2000)

Cell Regulatory Networks Important in Cancer Cells

Hanahan and Weinberg, Cell 144:646 (2011)

Cell cycle and its control

Cells must be able to proliferate

- during development- wound healing- stem cells in blood, small intestine, immune system

For cells to copy themselves they need to:

- Grow; make more stuff; e.g. proteins, lipids

- Copy their genetic material

- Segregate contents to daughter cells, especially…

- Segregate replicated chromosomes to daughter cells

Figure 8.3b The Biology of Cancer (© Garland Science 2007)

STARTor

Restriction Point

INTERPHASE:G1 + S + G2

Cell Cell CCycle ycle CommandmentsCommandments

A cell must replicate A cell must replicate every DNA sequence once, and only once, every DNA sequence once, and only once, during each cell division.during each cell division.

A cell must not start to replicate DNA unless its mass is sufficieA cell must not start to replicate DNA unless its mass is sufficient to support nt to support cell division.cell division.

If If the DNA is damaged, a cell must repair the damage before cell the DNA is damaged, a cell must repair the damage before cell division.division.

A cell must not divide until DNA replication has been completed.A cell must not divide until DNA replication has been completed.

Each cell must receive a complete complement of replicated DNA.Each cell must receive a complete complement of replicated DNA.

A cell resting in quiescence (GA cell resting in quiescence (G00) must not reenter the cycle u) must not reenter the cycle u

nless a proper mitogenic signal is receivednless a proper mitogenic signal is received..

A terminalA terminally differentiated cell must not reenter the cycle.ly differentiated cell must not reenter the cycle.

Figure 8.6 The Biology of Cancer (© Garland Science 2007)

The Restriction Point: Integrating GO : NO-GO Signals

Figure 8.1 The Biology of Cancer (© Garland Science 2007)

The Restriction Point: Integrating GO : NO-GO Signals

Two Two types types of genes are mutated in cancerof genes are mutated in cancer::

Loss of cell cycle control at the Restriction PointLoss of cell cycle control at the Restriction Point

tumor suppressorstumor suppressors

proto-oncogenesproto-oncogenesActivity: stimulate cell cycle progression

Mutation in cancer: gain of functionproto-oncogene = wt; oncogene = mutant

Examples: cyclin D1, Mdm2, myc, ras

Activity: Inhibit cell cycle progressionMutation in cancer: loss of functionExamples: Rb, p53, p16, ARF, PTEN

GG00

MM

G2G2

SS

G1G1

Figure 8.4 The Biology of Cancer (© Garland Science 2007)

Cell Cycle CheckpointsThe Guardian Mechanisms of

the Genome

THEY ARE DISRUPTED IN CANCER!

S Phase of the Cell Cycle

MCMHelicase

During the S phase, the duplicated DNA is rearranged through cohesion to form two sister-chromatids attached to each other by cohesins

The cohesins will be removed during mitosis to allow sister-chromatid separation

S Phase of the Cell Cycle

Mitosis

Figure 8.3a The Biology of Cancer (© Garland Science 2007)

Mitosis in NewtLung Cells

blue = DNAgreen = microtubules

At the end of the day:You need to do metaphase correctly

This requires organizing microtubules….

Kerry Bloom

Kinetochore

Microtubule

Microtubule

KinetochoreCentromere

Ted Salmon

and attaching them to kinetochores.

The Metaphase to Anaphase Transition:The key step during mitosis

Metaphase to anaphase transition in a plant cell

Figure 8.3b The Biology of Cancer (© Garland Science 2007)

STARTor

Restriction Point

INTERPHASE:G1 + S + G2

G1-S and G2-M are the major control points in the cell cycle

Rao and Johnsoncell fusion experiments

1. Fuse M phase cell with interphase cell: Interphase nucleus enters M

2. Fuse S phase cell with G1 cell: The G1 nucleus enters S phase

3. Fuse S phase cell with G2 cell: The G2 nucleus does not enter S phase

Cyclin Dependent Kinases Regulate the Cell Cycle

Phosphorylation of CDK Targets Changes Their Activity

Now performsa cell cycle function

Experimental Systems Important for Cell Cycle Studies

Arbacia punctulata

Xenopus laevisSchizosaccharomyces pombe

Saccharomyces cerevisiae

Budding Yeast Saccharomyces cerevisiae

Lee Hartwell

Hartwell was interested in the protein synthesis machinery

Budding Yeast: a genetic eukaryotic model organism

Let’s look for mutants that cannot synthesize proteins

Isolating Temperature Sensitive Mutants in Haploid Yeast

Lee Hartwell

Serendipity: a scientist’s best friend!

Brian Reid, an undergrad, needs to look at a microscope to follow a mutant. They realize that bud size stores information about the cell cycle

Brian Reid

Permissive (low) temperature Restrictive (high) temperature

Cdc Mutants Arrest at the Same Cell Cycle Phase

cdc mutant growingat permissive temp (23C)

cdc mutant growth arrested after 6 hrs at non-permissive temp (36C)

The Behavior of a Temperature Sensitive cdc Mutant

How to Clone cdc Genes in Yeast

Cdc Genes Encode Proteins Needed for DNA ReplicationStudies in S. cerevisiae

Fission yeast: Schizosaccharomyces pombe

Cdc Genes Encode Proteins Needed for the G2-M Transition:Studies in S. pombe

cdc2+ encodes a kinase Moreover = cdc28 in S. cerevisiae!

And they can substitute for one another!!!

Sir Paul Nurse

START (Restriction Point)Cdc2 (fission)

Cdc28 (budding)

Cdc2 (fission)Cdc28 (budding)

This is all great and

yeast are really cute and interesting, but

Can we really learn something from all of this about humans?

Schizosaccharomyces pombe

Sir Paul Nurse

Crazy idea

Let’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library

It worked for us with budding yeast genes. Why not try human genes?

Human cdc2 rescues cdc2 mutants!!

Elongated cdc2 mutants, failing to undergo mitosis

cdc2 mutants, complemented by a

human cdc2 gene

Melanie Lee

Summary

- A genetic approach in fission and budding yeasts reveals genes that are essential in promoting the cells through the cell cycle

- Key genes encode a protein kinase called CDKs for Cyclin-Dependent Kinases

CDK1 = the protein encoded by cdc2/CDC28

What about cyclins? How were they discovered?

Tim Hunt

Woods Hole Marine Biological Laboratory

Cyclin was Discovered in Sea Urchin Embryos

ProteinLevel

Time

cyclin A cyclin B

M M M

can stimulate to

lay lots of eggs

mitosis mitosis mitosis

(Spisula is actually a clam.)

OK, but what does this have to do with CDK’s??

I have theanswer!

Overview of the frog life cycle

1 mm

spermtadpole feeds, grows

and becomes an adult frog

OOCYTE GROWS WITHOUT DIVIDING(MONTHS) FERTILIZATION

FERTILIZED EGG DIVIDES WITHOUT GROWING(HOURS)

The Maturation of Frog Eggs

The Maturation of Frog Eggs

An Assay for Maturation Promoting Factor (MPF)

Yoshio Masui, 1971

MPF Activity Peaks Before Each Cell Division

Moreover, MPF has kinase activity

Purification of MPF: The Birthof Cyclin Dependent Kinases

This is cdc2+!!(Cdc28 in

S. cerevisiae)

This is cyclin!!

Which = cdc13+

in S. pombe

Phosphorylation of CDK Targets Changes Their Activity

Now performsa cell cycle function

The Nobel Prize in Physiology or Medicine, 2001

“for their discovery of key regulators of the cell cycle”