GE636: Cell Cycle Regulation:

Growth Factors, Oncogenes and Tumor Suppressor Genes,

A graduate-level course offered by Bruno Calabretta, MD, PhD, and other faculty at

Kimmel Cancer Center

Thomas Jefferson University,

Wednesdays, 2-4 pm, BLSB rm 602

Fall, 2002
 
 

Yeast Cell Cycle and Cell Cycle Checkpoint Lectures

September 11 and 18, 2002

Charles Brenner, PhD

charles.brenner@dartmouth.edu

Version 1.5.  For the latest update, please reload http://dartmouth.edu/~brenner/ge636.html

In the first two weeks of GE636, we will introduce some of the key cell cycle concepts, focusing on the yeast cell cycle and cell cycle checkpoints.

0.  The reading, without which no student should begin this course, is Chapter 17 of Molecular Biology of the Cell, 3rd Edition, by Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and James D. Watson.  Those students that have not taken Genetics 611 (or need a refresher) should also study our introductory Yeast Genetics lectures.

 
Alberts, Bray, Lewis, Raff, Roberts and Watson crossing Abbey Road

During several of the weeks of this course, students will present an outstanding paper or two, chosen by the instructor du semaine.  

Reading these notes and reading the text are not a substitute for reading the assigned papers and the primary literature referenced in the text and in the notes.  Do talk among yourselves and with faculty and other scientists in order to bring the sharpest, most inquisitive, and most informed discussion to the thirteen classes.  These notes provide an outline of the material to be covered in the first two weeks--please bring them to class.

The semester's schedule is as follows:

Week

Date

           Topic

  Professor

1

September 11

The yeast cell cycle

Brenner

2

September 18

Cell cycle checkpoint

Brenner

3

September 25

Cyclins, Cdks and Cdk inhibitors

Calabretta

4

October 2

E2F transcription factors and RB family genes

Calabretta

5

October 9

Regulation of G2/M transition

Donovan

6

October 16

p53 in cell cycle regulation

Mercer

7

October 23

TGF-b signaling

Mercer

8

October 30

Cell cycle regulation by IGF-1

Baserga

9

November 6

Cell cycle regulation by hemopoietic cytokines

Calabretta

10

November 13

Role of telomerase in immortalization and senescence

Mercer

11

November 20

Oncogenic receptor tyrosine kinases:  the EGF receptor

Wong

12

November 27

Oncogenic nonreceptor tyrosine kinases: BCR/ABL

Calabretta

13

December 4

The MYC oncogene

Calabretta

 

 

 

 

 

 

 

1.  Cells alternate between Mitosis and Interphase. At the light microscope level, the singular hallmark event of the cell cycle is mitosis and all cells can be divided into those that are going through mitosis and those that are not going through mitosis. Thus, the most fundamental cell cycle definition is that between M phase and Interphase. The salient features of mitosis are


 


 


 


 

2.  The most conspicuous feature of Interphase is chromosomal DNA replication, also known as S phase.

3.  Embryonic cell cycles alternate between rapid S phase and M phase without growth and can complete cell division cycles in a few minutes. In addition to making and segregating sister chromosomes, embryonic cells are also subdividing and organizing the fertilized oocyte into a multicellular body plan. Embryonic cells are a good example of how there can be cell division without growth.

4.  Differentiated cell-division cycles almost always involve growth. Such cells use environmental signals to coordinate growth with division. Differentiated cells are not fully occupied with S phase and M phase and spend most of their time prior to commitment to S phase, in G1, or prior to commitment to M phase, in G2. In G1, a liver cell can be a liver cell and brain cell be a brain cell. In G2, unfertilized oocytes remain in stand-by mode for decades. There are, obviously, many examples of terminally differentiated cells that undergo few or no cell divisions. Neurons are good examples of how there can be growth without division.

5.  Because cells spend so much time in G1 and/or G2 doing cell-type specific functions, much of cell division control is devoted to mechanisms devoted to not committing to S phase or not committing to M phase. Typically, growth factors are required to commit cells to S phase. Oncogenic mutations and losses of tumor suppressor genes frequently override growth factor requirements and other cell division controls.

6.  The yeast, Saccharomyces cerevisiae, is a single-celled fungus which exercises developmental options at the G1 stage of its cell division cycle.  Starvation of yeast for nutrients arrests them as small, unbudded cells in early G1 phase. Addition of nutrients to early G1-arrested cells allows them to begin growing. If haploid cells achieve a critical size and are not exposed to mating pheromone, they commit to a complete cell cycle. Bud emergence corresponds to the time of S phase initiation.

However, if haploid cells are exposed to cells of the opposite mating type, they arrest in late G1 phase, directing their growth toward the source of mating pheromone in a developmental process known as "shmooing."

Thus, at G1 phase, nutrients can be seen as a positive growth and division factor and mating pheromone can be seen as a negative and overriding cell-division factor. The execution point at late G1 phase that depends on cell size and absence of pheromone is called START.

A large number of mutants that are temperature-sensitive for mating (ste for sterile) and temperature-sensitive for cell division (cdc for cell-division cycle) were obtained before G1 control could be understood at a molecular level. One of the keys to the mating versus cell-division decision is Far1.

Budding Yeast Life Cycle and Cell Cycle
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

7.  Some cell division processes are dependent on other cell division processes. Lee Hartwell was the first to screen for recessive budding yeast mutants that are temperature sensitive for growth and arrest with characteristic cell division morphologies. The morphologies of cdc mutants arrest are called terminal phenotypes. It is important to appreciate that the appearance of cells at the terminal phenotype does not necessarily represent the state of the cell at the execution point. While many cell-cycle events depend on other events, at least three independent cell cycles can be described in yeast, the cytoplasmic cycle, the chromosome cycle, and the centrosome cycle. Each of these cycles, wheels within the wheel, depends on START for initiation and each must be completed for cells to undergo the metaphase to anaphase transition that marks the denouement of mitosis.  Here are some examples:


 


 

We will return to the matter of dependent cell-cycle processes later to examine which may illustrate simple substrate-product relationships and which appear to indicate true cell-cycle checkpoints.

8.  The major cell cycle control step in most well characterized eukaryotic cells is controlled by a multiprotein complex that includes homologs of Cdc28, cyclins and other proteins.  As we pointed out earlier, budding yeast cdc28 mutants are arrested at START in late G1 phase, which is the principal restriction point of the yeast cell division cycle.  Fission yeast and vertebrate and invertebrate oocytes are arrested at G2/M with inactive forms of the orthologous proteins.  Mammalian somatic cells in G0/G1 arrest also have inactive forms of the same protein complexes.  This story emerged from many lines of experimentation leaving us with many different names for similar protein kinase complexes:
 

Maturation or M-Phase Promoting Factor

Cyclin-Dependent Kinase

Proline-Directed Kinase

p34

Growth-Associated Histone H1 Kinase Activity

Cdc28-Cln and Cdc28-Clb Complex

cdc2-cdc13 Complex
 
 

9.  In S. cerevisiae, Cdc28 is the only Cyclin-Dependent Kinase catalytic subunit that promotes cell cycle transitions at G1 and G2.  Cdc28 activity is regulated by nine cyclins that accumulate, for the most part, at START and at G2/M that are regulated transcriptionally and proteolytically, several protein kinases and phosphoprotein phosphatases, and at least four other binding proteins.  For an introduction to the vast literature on Cdc28 regulation, see the Proteome entry.

10.  In yeast, getting mutants that uncouple normally coupled processes is a key to understanding complexity and specificity.  Before Far1, there were many yeast mutants that were deficient in signal transduction in response to pheromone but they all blocked pheromone-induced transcriptional activity and cell-cycle arrest in response to pheromone.  Thus,  it was possible to view mating pheromone-induced signal transduction as a process that depended totally on transcriptional reprogramming of the cell cycle.  One could view the function of the G-protein coupled-receptor/MAP kinase cascade as turning on "immediate early genes" which would be important for the synthesis of early, middle and late gene products.  Because it was possible to get a far (factor arrest) mutant that induced pheromone-mediated transcription but did not arrest the cell cycle, a component of the cell cycle arrest fork in the signal transduction cascade could be studied.  Chang and Herskowitz provided genetic evidence for Far1 inhibiting the Cdc28-Cln2 complex, which was early evidence for the existence of inhibitors of specific CDK-cyclin complexes.

11.  Checkpoints:  What is true for phage may not be true for the elephant.  Reading an interview with Lee Hartwell reminds one that the attempt to explain the function of cell cycle genes in yeast and other systems was predicated on earlier work on phage morphogenesis.  In phage there are many examples of late events that cannot take place without completion of earlier events.  In yeast and in mammalian cell cycles, there are some events that appear to follow the "domino theory" of total dependence on prior events and there are other processes that are not directly coupled.  Work summarized in point 7 above illustrated that there are at least three sets of dependent cellular cycles that are independent of each other.  The cytoplasmic cycle, chromosome cycle and the centrosome cycle all depend on START and all must be completed for the metaphase to anaphase transition to occur.  Might it be possible to explain the premitotic terminal phenotypes of cell cycle mutants with the principles of phage morphogenesis, i.e., that cell cycle delays in response to noncompleted events are solely due to the inavailability of cellular substrates for mitotic products?

It is a fact that yeast cells arrest prior to mitosis in response to DNA damaging agents.  Is this fact, by itself, sufficient to invoke the concept of a cell-cycle checkpoint?  Principles from phage morphogenesis and Occam's Razor would hold that damaged DNA might not be an adequate substrate for mitosis.  However, if drugs or mutants could be found that were to allow damaged DNA to proceed through mitosis, then it would become more difficult to argue that damaged DNA is structurally unfit to participate in mitotic processses.  P.N. Rao's group and others had shown that treatment with caffeine or fusion with mitotic cells would cause cells with damaged DNA to prematurely condense their chromosomes.  One way to accommodate the caffeine result within a morphogenetic paradigm was to hypothesize that caffeine binds to damaged DNA, allowing it to "pass" as an undamaged DNA substrate.  Another, suddenly simpler, hypothesis is that caffeine inactivates an inhibitor of cell cycle progression that prevents cells with damaged DNA from entering mitosis.

When Arthur Pardee's group showed that caffeine enhances lethality due to DNA damaging agents, the stage was set for someone to show that a specific gene product is required for the DNA damage checkpoint and that its loss confered radiation sensitivity, not due to the inability to repair lesions per se but due to the failure to detect those lesions and/or delay mitosis for their repair.  This was the backdrop for Ted Weinert's screen for yeast rad mutants that fail to arrest in G2 in response to X-rays.

12.  Signal transduction pathways that enforce cell-cycle dependencies and prevent mitotic catastrophe.  In Molecular and Cellular Biology of the Yeast Saccharomyces, volume 3, Lew, Weinert and Pringle review yeast cell cycle control and make a variety of critical observations.  First, they restrict use of the term "checkpoint controls" to signify signal transduction pathways that delay progression through the cell cycle in response to perturbation of specific events.  Thus, they do not refer to cell cycle boundaries (G1/S or G2/M, for example) as checkpoints and they do not refer to all negative regulators of the cell cycle as checkpoints.  Which cellular perturbations cause delays that depend on specific signal transduction pathways?  The following is an incomplete list of known checkpoint pathways in yeast.
 

¬…       inhibition of DNA replication.  Lew and co-authors review data suggesting that DNA replication forks themselves may function as a replication signaling complex whose presence blocks mitotis and that Mec1 and Rad53 are effector proteins for this checkpoint.


 

¬…       damage to DNA.  rad9, rad17, rad24 and mec3 appear to be required to transduce DNA damage signals through Mec1 and Rad53 effectors.


 

¬…       problems assembling the mitotic spindle.  mad1,2 and 3 and bub1, 2 and 3 fail to arrest in response to microtubule-directed drugs.  Execution of the spindle checkpoint pathway requires Pds1.

13.   What do you think would be the consequences of loss of function mutations in the human homologs of MEC1 and TEL1?

 

14.  Reading for September 18, 2002:

Sanchez et al, 1999 (full text) and Schwartz et al, 2002 (full text).

 

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