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 Hanahan and Weinberg, Cell 144:646 (2011)

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

INTERPHASE: G1 + S + G2 START or Restriction Point Figure 8.3b The Biology of Cancer (© Garland Science 2007)

Cell Cycle Commandments A cell resting in quiescence (G0) must not reenter the cycle unless a proper mitogenic signal is received. A terminally differentiated cell must not reenter the cycle. A cell must not start to replicate DNA unless its mass is sufficient to support cell division. A cell must replicate every DNA sequence once, and only once, during each cell division. If the DNA is damaged, a cell must repair the damage before cell division. A cell must not divide until DNA replication has been completed. Each cell must receive a complete complement of replicated DNA.

The Restriction Point: Integrating GO : NO-GO Signals 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)

Loss of cell cycle control at the Restriction Point Two types of genes are mutated in cancer: G0 M G2 S G1 proto-oncogenes Activity: stimulate cell cycle progression Mutation in cancer: gain of function proto-oncogene = wt; oncogene = mutant Examples: cyclin D1, Mdm2, myc, ras tumor suppressors Activity: Inhibit cell cycle progression Mutation in cancer: loss of function Examples: Rb, p53, p16, ARF, PTEN

Cell Cycle Checkpoints The Guardian Mechanisms of the Genome THEY ARE DISRUPTED IN CANCER! Figure 8.4 The Biology of Cancer (© Garland Science 2007)

S Phase of the Cell Cycle MCM Helicase

S Phase of the Cell Cycle 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

Mitosis

Mitosis in Newt Lung Cells blue = DNA green = microtubules Figure 8.3a The Biology of Cancer (© Garland Science 2007)

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

This requires organizing microtubules….

and attaching them to kinetochores. Centromere Kinetochore Microtubule Kerry Bloom Ted Salmon Kinetochore Microtubule

The Metaphase to Anaphase Transition: The key step during mitosis Metaphase to anaphase transition in a plant cell

INTERPHASE: G1 + S + G2 START or Restriction Point Figure 8.3b The Biology of Cancer (© Garland Science 2007)

G1-S and G2-M are the major control points in the cell cycle Fuse M phase cell with interphase cell: Interphase nucleus enters M Fuse S phase cell with G1 cell: The G1 nucleus enters S phase Fuse S phase cell with G2 cell: The G2 nucleus does not enter S phase Rao and Johnson cell fusion experiments

Cyclin Dependent Kinases Regulate the Cell Cycle

Phosphorylation of CDK Targets Changes Their Activity Now performs a cell cycle function

Experimental Systems Important for Cell Cycle Studies Saccharomyces cerevisiae Arbacia punctulata Schizosaccharomyces pombe Xenopus laevis

Budding Yeast Saccharomyces cerevisiae

Budding Yeast: a genetic eukaryotic model organism Hartwell was interested in the protein synthesis machinery Lee Hartwell Let’s look for mutants that cannot synthesize proteins

Isolating Temperature Sensitive Mutants in Haploid Yeast

Serendipity: a scientist’s best friend! Lee Hartwell Brian Reid 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

Cdc Mutants Arrest at the Same Cell Cycle Phase Permissive (low) temperature Restrictive (high) temperature

The Behavior of a Temperature Sensitive cdc Mutant cdc mutant growing at permissive temp (23C) cdc mutant growth arrested after 6 hrs at non-permissive temp (36C)

How to Clone cdc Genes in Yeast

Cdc Genes Encode Proteins Needed for DNA Replication Studies in S. cerevisiae

Fission yeast: Schizosaccharomyces pombe

Cdc Genes Encode Proteins Needed for the G2-M Transition: Studies in S. pombe Sir Paul Nurse cdc2+ encodes a kinase Moreover = cdc28 in S. cerevisiae! And they can substitute for one another!!!

START (Restriction Point) Cdc2 (fission) Cdc28 (budding) START (Restriction Point) Cdc2 (fission) Cdc28 (budding)

yeast are really cute and interesting, but 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

Crazy idea It worked for us with budding yeast genes. Why not try human genes? Sir Paul Nurse Let’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library

Human cdc2 rescues cdc2 mutants!! Melanie Lee cdc2 mutants, complemented by a human cdc2 gene Elongated cdc2 mutants, failing to undergo mitosis

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?

Woods Hole Marine Biological Laboratory Tim Hunt

Cyclin was Discovered in Sea Urchin Embryos can stimulate to lay lots of eggs Protein Level Time cyclin A cyclin B M

(Spisula is actually a clam.) mitosis (Spisula is actually a clam.)

OK, but what does this have to do with CDK’s?? I have the answer!

Overview of the frog life cycle OOCYTE GROWS WITHOUT DIVIDING (MONTHS) FERTILIZED EGG DIVIDES WITHOUT GROWING (HOURS) FERTILIZATION 1 mm sperm tadpole feeds, grows and becomes an adult frog

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 Birth of 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 performs a cell cycle function

The Nobel Prize in Physiology or Medicine, 2001 “for their discovery of key regulators of the cell cycle”