1. The Hallmarks of Cancer
Hanahan and Weinberg, Cell 144:646 (2011)

2. Newer Hallmarks of Cancer
Hanahan and Weinberg, Cell 144:646 (2011)

3. Therapeutic Targeting of the Hallmarks of Cancer
Hanahan and Weinberg, Cell 144:646 (2011)

4. Cell Regulatory Networks Important in Cancer Cells
Hanahan and Weinberg, Cell 100:57-70 (2000)

5. Cell Regulatory Networks Important in Cancer Cells
Hanahan and Weinberg, Cell 144:646 (2011)

6. Cell cycle and its control

7. 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

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

9. 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.

10. The Restriction Point: Integrating GO : NO-GO Signals
Figure 8.6 The Biology of Cancer (© Garland Science 2007)

11. The Restriction Point: Integrating GO : NO-GO Signals
Figure 8.1 The Biology of Cancer (© Garland Science 2007)

12. 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

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

14. S Phase of the Cell Cycle
MCM
Helicase

15. 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

16. Mitosis

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

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

19. This requires organizing microtubules….

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

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

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

23. 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

24. Cyclin Dependent Kinases Regulate the Cell Cycle

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

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

27. Budding Yeast Saccharomyces cerevisiae

28. 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

29. Isolating Temperature Sensitive Mutants in Haploid Yeast

30. 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

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

32. 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)

33. How to Clone cdc Genes in Yeast

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

35. Fission yeast: Schizosaccharomyces pombe

36. 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!!!

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

38. 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

39. 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

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

41. 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?

42. Woods Hole Marine Biological Laboratory
Tim Hunt

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

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

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

46. 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

47. The Maturation of Frog Eggs

48. The Maturation of Frog Eggs
An Assay for Maturation Promoting Factor (MPF)
Yoshio Masui, 1971

49. MPF Activity Peaks Before Each Cell Division
Moreover, MPF has kinase activity

51. Purification of MPF: The Birth of Cyclin Dependent Kinases
This is cdc2+!!
(Cdc28 in
S. cerevisiae)
This is cyclin!!
Which = cdc13+
in S. pombe

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

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