C2006/F2402 '11 OUTLINE OF LECTURE #25

(c) 2011 Dr. Deborah Mowshowitz, Columbia University, New York, NY.
Last update 05/03/2011 09:12 AM .

Handouts: 25A -- Cell Cycle & Checkpoints
                 25B -- TK cascade, role of cyclin. 
This is an optional lecture. This material will NOT be on the final. If you want to enrich your understanding of the subject, see problem set 15.
I. Regulation of the (Normal) Cell cycle  

    A. Why does it matter? Why bring it up now? To understand what is wrong with cancer cells, we need to look at the normal cell cycle, and then how it goes wrong in tumor cells.

    B. Overview of Cell Cycle (Sadava fig. 11.3 (9.3) or Becker fig. 19-1.) See handout 25A.

1. Steps. G-1, S etc.; histones made in S too along with DNA.

2. Checkpoints

a. What's a checkpoint? A decision point in the cell cycle. Cells require certain internal and/or external factors to proceed past each checkpoint. If a particular process has not been completed successfully, or certain factors are not present, the cells cannot proceed past the corresponding checkpoint. Checkpoints are detected because cells 'hang up' or get stuck at these particular points in the cell cycle.

b. G1/S Checkpoint in mammalian cells

(1). Location in cycle: Actual checkpoint is in G-1, near end; determines whether cell will enter G-0 (non dividing or non cycling state) or S (or pause in G-1 and await further instructions). Irreversible decision is  to proceed past checkpoint and enter S.

(2). Significance: This is the major checkpoint/decision point for animal cells in the adult. 

(3). Role of GFs: Many growth factors (like EGF) act at this point -- signals from neighboring cells (GFs) are needed to enter S.

(4). Terminology: Also called "start' in yeast or "restriction point" in mammalian cells.

(5). Role in cancer: Most tumors have a defect in the switch that controls this checkpoint.

c. Other Checkpoints

(1). The other major checkpoint -- the G2/M Checkpoint (Border of G-2 & M). 

(a). Needed to check all components are ready for mitosis before proceeding. 

(b). This is the major checkpoint/decision point for cleaving eggs. (see Becker figs. 19-28 & 19-30) Cycle here is basically all S and M -- G1/S switch is in override.

(c). Note: Passage of this checkpoint in animal cells generally depends on the internal state of the cells, not on presence/absence of GF's.

(2). Additional checkpoints exist.   See texts (especially more advanced ones) if you are interested in more details and examples. 

    C. What controls Checkpoints?

1. Is regulatory protein an inhibitor or an activator? Fusions (between cells at different points in the cell cycle) imply decisions at checkpoints are controlled primarily by an "on switch" not release of  an "off switch." See Becker Fig. 19-32 or Sadava fig. 11.4 (9.4). You need presence of an activator signal (not loss of an inhibitor) to proceed past a checkpoint. 

2. Switch controlled by, or is, a protein kinase -- at least 2 different ones -- one for "start" and one for G2/M. In each case you need to have an active protein kinase. Production/activation of the switch protein complex involves a complicated process which can involve multiple inhibitors and/or activators, as explained below.

        3. What do checkpoint protein kinases do?

a. Allow cells to pass checkpoint and enter next phase of cell cycle

b. Different kinase needed to pass each checkpoint.

(1). Start kinase used for G1/S checkpoint

(2). MPF needed to pass G2/M checkpoint.

c. Each kinase phosphorylates a specific set of proteins (See handout 25A). What does phosphorylation achieve?

(1). May activate proteins needed to successfully complete next stage of cycle -- start DNA replication (for G1/S), disassemble nuclear membrane (for G2/M), separate chromosomes, etc. (see table on handout 25A)

(2). May inhibit proteins blocking the cycle -- for example, phosphorylation of rb releases E2F (a TF) to allow transcription. (See handout 25B, and more details below.)

4. Composition of Checkpoint kinases (See Sadava fig. 11.5 (9.5))

a. Each checkpoint kinase has 2 parts -- Cyclin and CDK.

b. CDK = cyclin dependent kinase (called p34, cdc2 etc.).

(1). This is the actual catalytic protein. Inactive without cyclin; therefore catalytic activity (not amount) of the kinase is dependent on cyclin (& other factors; see below).

(2). Stable protein. Level of kinase protein itself  remains steady in cycling cells. (Probably degraded if cells stay in G0 = exit the cell cycle.) 

c. Cyclin 

(1). Unstable protein. Builds up, peaks, degraded (in proteasome), repeats (See Becker fig. 19-34  or handout 25A for graph).

(2). Acts as an activator of the CDK. Cyclin is necessary, but not sufficient, to activate the CDK.

(3). Different cyclins for G1/S and G2/M; usually different CDK's too.

(4). Cyclin levels are regulated by controlling production & degradation of both cyclin and its mRNA.

d. Activation of checkpoint kinase is required. (See 25B).

(1). Complex of CDK + cyclin forms, but is inactive.

(2). Action of right combo of (additional) kinases and phosphatases active form. (See Becker fig. 19-35 if you are curious about the details)

(3). Activation is blocked by p53 (a protein that is inactive in many tumors) when DNA is badly damaged or DNA replication is blocked. (More details on p53 are below.)

   D. Overview of Events required to pass G1/S checkpoint -- See handout 25B.

1. Steps (See handout 25B)

a. Part 1 -- Synthesis of cyclin

  • GF binds to TK receptor. (See II below for how TK receptors work.)
  • Activated TK receptor triggers TK cascade. (ras is part of cascade.)
  • Last enzyme in cascade activates a TF.
  • Cyclin gene is transcribed, cyclin made. 

b. Part II -- Action & Fate of cyclin

  • Cyclin combines with preexisting proteins to assemble inactive kinase complex.
  • Kinase Complex (start kinase) is activated. (p53 blocks this step)
  • Start kinase phosphorylates target proteins (usually TFs or their inhibitors)
  • Phosphorylated target proteins enable cell to enter S (see role of rb & E2F on handout or below).
  • After checkpoint passed: Kinase complex disassociates, cyclin degraded.

2. Why so many multiple controls and steps?  If adult cell divides when it shouldn't cancer; if fails to divide get loss of repair (no healing) and degeneration in adult. So need division to be controlled very carefully . So have multiple "brake" and "accelerator" proteins in this system. Many cancers traced to loss of "brake protein" (like p53 or rb) or over production/activation of "accelerator protein" (like ras) see below.

    E. What is wrong with cancer cells? Usually multiple things.

1. G1-S switch is defective. (We will concentrate on this defect.)

a. Defective switch allows cell to multiply regardless of external signals.

b. Cell does not pause normally at the usual decision point --  proceeds too readily from G1 to S.

(1) Doesn't need positive signals (such as GF) in order to grow.

(2) Doesn't heed negative signals (such as p53) to stop growing.

c. Cycle is normal length (not shorter).

2. Additional mutations usually required. Additional mutations can do any or all of the following:

a. Contribute to unregulated growth 

b. Allow the cells to metastasize (spread to other locations).

c. Promote angiogenesis -- formation of blood vessels to feed the tumor

d. Allow cells to evade apoptosis (programmed cell death = cell suicide).

e. Turn on telomerase -- to avoid shortening of chromosomes at DNA replication

3. Certain genes are defective in many cancers. These include ras, rb, E2F, p53 & genes for TK receptors. Why these? See below.

II. An Aside: Signaling with RTK's. How do RTK's Work? 

    A. Importance of Catalytic Receptors

1. What are they? Catalytic Receptors are surface receptors whose intracellular domain is an enzymatic domain (or binds to one)

2. How do they work? Ligand binding activates the enzymatic domain. The active enzyme modifies a cellular protein, which binds to or modifies other proteins, etc.

3. Types: The enzymatic domain is usually a kinase, often a tyrosine kinase. We will stick to receptor tyrosine kinases & TK linked receptors.

4. What signaling molecules use TK receptors?

5. Results: Activation of TK receptors often leads to changes in TF's and transcription. (Note this not the usual case with G-linked receptors -- their end target is usually not a TF.)

    B. Important Properties of Receptor TKs

1. Receptor is usually a single pass protein 

2. Ligand binding usually leads to dimerization of receptors (Sadava fig. 7.7 (15.6) or Becker 14-17). Why does it matter that TK receptor monomers (or any protein) must dimerize in order to act?

a. Function: If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.

b. Inheritance: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-20 & 14-21 (14-19 & 14-20). Dimers form, but they are inactive. (These mutations are called 'dominant negatives.' Most negative, or lack of function, mutations are recessive.)

3. Dimerization activates a TK in cytoplasmic domain of receptor or in separate TK (that binds to receptor).

4. Active kinase auto-phosphorylates itself. TK adds phosphates (from ATP) to its own tyrosines -- each subunit phosphorylates the others.

5. Other proteins are activated by binding to phosphorylated TK's. (Sadava fig. 7.12 (15.10), or Becker 14-18)

a. Direct Effect: Some proteins can be activated by binding directly to TK.

  • Example? PLC. Ligand binding to TK-linked receptor can activate a type of PLC IP3 & DAG etc.
  • This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs. 
  • The cAMP pathway (as far as we know) can only be activated by GPCRs.  

b. Indirect Effect: Some proteins are activated by binding indirectly -- these proteins bind to "adapter proteins" that are bound to the TK. How it happens: 

(1). Adapter proteins bind directly to the  phosphorylated TK (to the phosphorylated tyrosines)

(2). Additional proteins bind to the adapters 

(3). Binding to adapters activation of the additional "recruited" proteins.

(4). Most famous (infamous?) example is the small G protein ras. Why does anyone care about ras?

(a). Overactive form of ras (stuck in the activated 'on' form) can cause cancer. Implies normal ras involved in control of cell cycle. (More on ras in lect. 25.)

(b). Large % (about 30%) of tumors have overactive ras.

c. SH2 domains. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains. 

d. Recruitment. Note that the initial target protein(s) to be activated come to or are "recruited by" the TK; this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.

III. Growth Factors, Growth Control, & Cancer -- Putting it All Together

This material is covered in Becker, chap 24, and Sadava sect. 11.7 (17.4) & the signaling chapters. We may not cover all of this, but it is all included as interesting background.

   A. How is the G1-S switch broken in cancer cells?

1. Problem can be at many different steps -- with production of CDK/cyclin complex, with its activation, &/or with its targets. (See handouts 25A & 25B)

2. Two major types of mutations responsible (See Sadava fig. 11.24 (17.15 & 17.17) -- these result in either

a. Stuck accelerator (stimulation uncontrolled) -- example: permanently active ras

b. Brake failure (inhibition fails) -- example: no p53

3. Types of mutations that cause cancer used to unravel normal G1/S signaling pathways & vice versa)

     B. Types of mutations that cause cancer

1. Can over-activate an activating (turn on) gene = "stuck accelerator" type of alteration -- activator is always 'on.'

a. Examples: Changes in genes for ras (so it's always in active form), or GF (always produced as autocrine), or GF receptor (always activated even without GF present), etc. See Sadava fig. 17.15 (8th ed).

b. Terminology:

(1). Normal activator gene is called a proto-oncogene

(2). Over-active version is called an oncogene. (See Becker table 24-1 for a list)

c. Oncogene can "over do it" 2 ways (see Becker fig. 24-15)

(1). Altered gene expression: Gene can be overexpressed  -- problem is with gene expression = level of mRNA production and/or translation = level of protein synthesis. Protein is normal, but you make too much of it (or you make it at the wrong time/place). Mutation is in regulatory sequence controlling gene, not in coding part. (ex: constitutive production of a GF)

(2). Altered gene product: Gene can be altered so protein made is constitutively active -- problem is with regulation of protein (not gene) activity -- protein is altered so it is always active. Mutation is in protein coding part of gene. Normal amount of protein is made, but protein is altered. (ex: production of altered GF receptor that is active without its ligand, or ras that is always active.)

2. Can inactivate a blocking gene = "brake failure" type of alteration -- inhibitor is always 'off.'

a. Terminology; Normal "brake" genes are called tumor suppressors (see Sadava fig. 17.17 (8th ed) for picture or Becker table 24-3 (24-2) for a list)

b. Examples: Most famous examples of tumor suppressors = rb and p53.

(1). p53 -- protein that causes block in cell cycle in damaged cells. Normal p53 acts as 'brake' on activation of CDK/cyclin complex. (see below & Becker fig. 19-39)

(2). rb -- one of the targets of start kinase. rb protein must be phosphorylated (and inactivated) for cell to enter S.  Rb 'holds up' the cycle until CDK/cyclin complex is properly activated. (See handout or below & Becker fig. 19-38). 

c. How mutations in tumor suppressors cause cancer: When p53 or rb (or other brake protein) is defective, cell keeps on growing when it should not -- in spite of damage (p53) or lack of growth factor signal (rb), etc. Result is often a tumor.

Try the problems in Set 15, part 1. Do them with a copy of handout 25B in front of you.

    C. How do Cancer Causing Mutations occur?

1. Somatic mutations can cause cancer (see Becker fig. 24-12)

a. What's a somatic mutation? A mistake in DNA replication that occurs in somatic cells, not in germ cells. Is not inherited or passed on to the next generation. Is passed on at mitosis to other somatic cells in the same person. Can be a point mutation (change in a few base pairs) or a rearrangement (deletion, insertion, inversion, translocation, etc. ).

b. Changes in Regulatory Sequences. Genes can become over-active [or inactive] because of rearrangements or other mutations that alter effects of enhancers, silencers, etc. Example of cancer caused by rearrangements of regulatory sequences: Burkitt's lymphoma. (Proto-oncogene is placed next to a very strong enhancer.) See Becker fig. 24-13.

c. Changes in Protein Coding Sequences. Protein can become over-active [or inactive] because of changes in the gene that alter the amino acid sequence of the protein. Example caused by rearrangement: chronic myelogenous leukemia (CML -- too many white blood cells) -- cells make a chimeric TK that is constitutively active. Activity of normal TK is regulated; activity of chimeric TK cannot be turned off. See Becker Fig. 24-14 & 24-15.

2. Viruses can cause cancer --  viral DNA integrates into normal cell DNA and brings in new genes (or messes up old ones of host).

a. Virus can carry in a new gene/protein -- an oncogene or a gene that codes for the inhibitor of a tumor suppressor. See Becker fig. 24-18. Ex: HPV (which can cause cervical cancer) makes two proteins that inhibit the tumor suppressors p53 and rb.

b. Virus DNA can destroy a host gene -- virus can integrate into DNA in middle of a normal gene and inactivate that gene -- can knock out a tumor suppressor.

c. Virus DNA can carry in a new regulatory sequence -- virus can integrate into DNA near a normal host gene and provide an enhancer or silencer that changes expression of that host gene -- can turn on a proto-oncogene.

d. Most cancers are not caused by viruses. A few types of cancer are associated with viruses (see Sadava Table 17.1 (8th ed) or fig. 11.24 (9th)) but even in these cases the viruses alone are usually not sufficient to cause the cancer. An example: Cervical cancer is largely viral in origin, in that HPV infection is usually involved. (See Sadava p. 209, 9th ed.)  However HPV infection is not sufficient to cause it. A vaccine has been developed recently to prevent HPV infection, and there is considerable controversy about the use of the vaccine. For more background information, go to http://www.cdc.gov/std/hpv/ . For news on the subject, try Medical News Today.

Note: there is some recent evidence that some cases of prostate cancer are associated with a viral infection. See also medpage for a reference to the original report.

3. You can inherit a predisposition to cancer, not the disease itself. How can you inherit a "predisposition" = high chance of getting cancer?

a. Genes that affect DNA replication &/or repair.  People who inherit versions of genes giving low repair or high mutability (caretaker mutations) tend to get cancer -- sooner or later, one of the random mutations that occurs is likely to mess up a proto-oncogene or tumor suppressor gene as above. Example: xeroderma pigmentosum, which carries a high risk of skin cancer because of defects in the genes for DNA repair. See Becker, box 24A & fig. 24A-1.

b. Tumor suppressor mutations. People who inherit one defective copy of a tumor suppressor gene are ok. However, if the second copy of the gene gets messed up in a cell., there will be no good copy of the tumor suppressor gene in that cell. If both copies of a tumor suppressor gene in one cell get inactivated or lost, cancer can result.  (This is the "two-hit" hypothesis for how mutated tumor suppressors cause cancer. See Sadava 17.16 (8th ed) or Becker fig. 24-17. Example: retinoblastoma, which causes a high risk of eye and ovarian tumors, is caused by defects in rb.

4. Most cancer is sporadic, due to somatic mutation. Not inherited.

5. Cancer develops in stages

a. Most cancers have more than one mutation

b. Selection -- selection for increasing loss of growth control occurs as disease progresses -- cells that grow more aggressively (due to additional mutations) outgrow the others.

c. Progression: Normal cell benign tumor malignant (invasive) metastasis (spreads) See Sadava 17.19 (8th ed) or Becker fig. 24-9 (24-10).

d. What sort of mutation causes cancer? Is cancer caused by a 'lack of function' mutation or 'gain of function' mutation? Usually both. Most cancer cells have multiple mutations. See Sadava 17.18 (8th ed.)

     D. How do Rb & p53 fit into normal cycle? How do mutant tumor suppressor genes/proteins cause cancer.

1. p53 -- most commonly mutated gene in human cancers. 

a. What normal p53 is/does: 

(1). p53 is a protein that is unstable; it only builds up if there is extensive DNA damage, for example, from irradiation or chemicals. (In the absence of damage,  p53 is degraded by the proteasome.)

(2). When p53 builds up, it causes either

(a). A temporary block in the cell cycle (until damage is repaired), or

(b). Apoptosis (programmed cell death) in irreversibly damaged cells. Cell "commits suicide" and dies without damaging neighbors. See Becker, figs. 14-27 & 14-28 (14-25 & 14-26).

(3). Normal p53 acts as "brake" on activation of CDK/cyclin start complex. (see handout 25 B, or Becker fig. 19-39.

(4). Not all DNA changes are detected by p53. Changes in base sequence that do not disrupt the 3D structure of DNA or interfere with DNA replication do NOT generate a 'damage signal' and do not affect p53 stability. Therefore p53 does NOT prevent all mutations, and if a mutation occurs (due to a mistake in DNA replication, etc.) p53 does not interfere with replication of the mutated cells (because their DNA is normal in overall structure even if it is changed in sequence).

b. Results of p53 failure: When p53 (or other brake protein) is defective, and there is extensive DNA damage, there is no block to DNA replication (or cell cycle), and damaged cell keeps on growing   cells with mutations tumors (sometimes).

2. Rb & E2F -- see Becker fig. 19-38. See diagram on  handout 25B for role of Rb and E2F.

a. Role of E2F: E2F = TF needed to make proteins to enter S.

b. Role of rb protein: inhibitor of E2F. Rb holds E2F in check until CDK/cyclin is properly activated by ras et al. 

c. Role of start kinase: Active cyclin/CDK complex (= start kinase) phosphorylates rb protein, inactivating it and releasing active E2F.

d. How inactive rb causes loss of growth control: If both copies of RB gene in a cell are knocked out, then cell makes no rb protein, and uncontrolled growth develops (because E2F cannot be inhibited.)

e. How role of rb found:   This discovered because individuals who inherited one defective copy of RB developed tumors. Cells in tumor had both copies of RB knocked out (tumor cells =  rb -/- = homozygous defective). Cells in other tissues were -/+ (heterozygous). See Sadava fig. 17.16 (8th ed) or signaling chapter (9th).

f. Led to discovery of importance of E2F

3. Rb vs p53: p53 regulates activation of CDK/cyclin complex. Rb is phosphorylated by active complex. So rb acts "downstream" of p53 = after p53, at a later step in the pathway. If rb is out of commission, it doesn't matter what p53 does -- p53 can't block the cycle.

Note FYI: the real situation is quite complex and there is some evidence that ras may also directly effect rb without cyclin. This complexity and/or uncertainty is reflected in your texts -- the different diagrams in the texts differ in where they put cyclin relative to rb/E2F. Hopefully, this matter will be cleared up shortly and the information we get will be useful in preventing growth of cancerous cells.

   E . What can we do to treat/cure cancer? What use is all this?

1. Classic methods. Usual methods depend on fact that cancer cells are actively dividing and most cells of adult are not. So cure/treatment is to try to remove as many cancerous cells as possible (by surgery) and then destroy any remaining cancer cells with drugs (chemotherapy) and/or radiation. This destroys any normal dividing cells as well, so it has serious side effects. (It also loses effectiveness with time because it selects for growth of drug/radiation resistant cells.)

2. New methods (mostly still under development). Target specific protein(s) in cancer cells or their supporting factors that allow unregulated growth or metastasis. See Becker Ch 24. Examples:

a. Small molecules that block enzymes/receptors:

Gleevec  (Imatinib)  -- inhibits a constitutive kinase; binds to and blocks the ATP binding site. (A review of Gleevec)

Tamoxifen -- binds to and inhibits an estrogen receptor.

Iressa (Gefitinib) -- binds to a GF receptor. 

b. Monoclonal antibodies (See Becker box 24B)

Herceptin & Erbitux are monoclonal antibodies to GF receptors on tumor cells; Selling Erbitux stock is what landed Martha Stewart in jail. (A sobering note: current prices for these drugs run from $3000 to over $9000 per month.)

Avastin is a monoclonal antibody to a VEGF -- a growth factor that promotes vascularization (growth of blood vessels to support the tumor). See Becker fig. 24-22 for effects of blocking vascularization.
For a recent use of Avastin (really a similar drug) see NY Times 4/27/10 on Eye Treatment for Vision Loss in Diabetics

How is the 'war on cancer' doing? See NYTimes, 4/09: In Long Drive to Cure Cancer, Advances Have Been Elusive.

Another report: from NY Times 4/28/10 A Study offers Clues on Therapy for Cancer

A more recent (& technical) update from Science Magazine: Exploring the Genomes of Cancer Cells: Progress and Promise. This comes from a special issue of Science (March 25, 2011) -- 'The Cancer Crusade at 40'.