C2006/F2402 '08 OUTLINE OF LECTURE #18

(c) 2008 Dr. Deborah Mowshowitz, Columbia University, New York, NY.
Last update 04/02/2008 05:17 PM .

Handouts 18A (Signaling with RTK's) & 18B (Regulation of Cell Cycle).  Not on web.

I. Signaling with Lipids (PI Derivatives)

    A. Many inositol derivatives are involved in signaling -- this is a current hot subject of investigation.

1. What is PI? Phosphatidyl inositol (PI) is a membrane lipid; inositol part faces cytoplasm.

2. Role of Kinases

a. Kinases add additional phosphates to hydroxyls on PI.

b. Many different products are possible, as PI has 5 free hydroxyls. Examples:

  • PIP2 or PI 4,5 bisphosphate-- has phosphate added to 4 & 5 positions.
  • PIP3  -- has phosphates on 3, 4, & 5 positions.

c. Activation of kinases and phosphatases (to remove the added phosphates) regulated by hormones, growth factors, etc.

3. What does phosphorylated PI do?

a. Recruitment: Phosphorylated PI (for example, PIP3) may recruit enzymes to the membrane; binding to the modified PI  activates the enzymes (no second messengers).

b. Generate second messengers: Phosphorylated PI (for example, PIP2) may be split to generate second messengers, such as IP3 and DAG. The second messengers bind to and activate enzymes.

   B. Details for DAG/IP3/Ca++ pathway. See handout 12A & Becker figs. 14-9 & 14-10 (10-8 & 10-9) or Sadava 15.13 (15.11)

1. How IP3 and DAG are generated

a. Activated G protein binds to phospholipase C (PLC)

b. PLC cleaves PIP2 in membrane → two parts. For structures see handout 12A and Becker fig. 14-9 (10-8).

(1). PIP3  -- soluble, in cytoplasm; also known as InsP3

(2). DAG = diacyl glycerol = glycerol with two fatty acids. DAG. remains in membrane.

2. Reminder: Role of IP3/Ca++

a. IP3 opens Ca++ channels in the ER, raising Ca++ in cytoplasm. (Becker fig. 14-11 (10-10) for IP3 effect; fig 10-11 for overall Ca++ regulation.)

b. Ca++ acts alone or as complex with calmodulin -- complex (calmodulin-Ca++) or Ca++ alone binds to and alters activity of many proteins. (See Becker fig. 14-13 (10-12)  for pictures of calmodulin.)

c.  Ca++ affects many processes -- sometimes called "3rd messenger." Changes in [Ca++] can trigger exocytosis (& secretion) or muscle contraction & big changes in Ca++ levels are involved in egg fertilization (see Becker fig.14-14 (10-13) or Sadava  15.14 (15.12) for some nice pictures). 

3. Role of DAG

a. DAG (in membrane) activates protein kinase C (= PKC, not to be confused with PLC).

b. "PKC" is a family of related enzymes involved in many different processes -- act by phosphorylating other proteins. If you are interested, see Becker or advanced texts for details. Some PKC's require Ca++.  PKC and PKA have different target proteins.

Try problems 6-5, 6-10 & 6-16.
 

II. Signaling with RTK's. How do RTK's Work? 

    A. Important Properties of Receptor TKs

1. Receptor is usually a single pass protein 

2. Ligand binding usually leads to dimerization of receptors (Sadava fig. 15.6 or Becker 14-17 (10-17).)

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. 15.10 (15.9), or Becker 14-18 (10-18)) See handout 18A.

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 

(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. (See below.)

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

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

d. Recruitment. Note that the target protein to be activated comes to or is "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.

6. What ligands use TK receptors?

Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors. Insulin, but not most other endocrines, acts through TK receptors. (See Sadava 15.6)

7. 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. A human example of TK receptor signaling:  FGF (Fibroblast growth factor) and FGF Receptor.  Significance of dimerization. See Becker figs.14-19 & 14-20 (10-19 & 10-20).

1. FGFR is a TK receptor  

2. FGF & FGFR needed for proper development as described in Becker chap.10. Failure of signal transmission (or premature transmission) causes developmental abnormalities. See Becker fig. 14-20 (10-20).

3. How can achondroplasia (a type of dwarfism), due to a defective FGF receptor, be dominant? See Becker fig. 14-19 (10-19), and box on handout 18A.

a. The general principle: TK receptor monomers must dimerize in order to act. If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.

b. An important consequence: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-19 & 14-20 (10-19 & 10-20). Dimers form, but they are inactive. (See dominant negatives, below.)

c. This case: In a heterozygote for achondroplasia, 1/2 the FGF receptor (monomers) are defective, therefore dimers that form are defective.

(1). In the example shown in Becker & on handout,  the cytoplasmic domain of the defective receptors is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. (Fig. 14-19) Therefore the signaling is badly disrupted. (In the example shown in Fig. 14-20, the FGF is needed to turn on formation of certain embryonic tissues, so the mutant fails to form these tissues. This type of mutation is called a 'dominant negative' as explained below.)

(2). (FYI) In most cases of human achondroplasia, the molecular explanation is different. In these cases, the mutation is in the transmembrane domain of FGF3 Receptor and dimers form, but act abnormally. (In these cases, the FGF signal is needed to turn on bone differentiation and turn off cell growth. Mutant dimers signal prematurely, so differentiation starts -- and cell growth stops -- before bones are long enough. This type of mutation is called a 'gain of function' mutation, because it works when it shouldn't.)

4. Dominant Negatives -- Some Additional Background

a. Definition: An abnormal allele like the one that produces the FGF receptor without a cytoplasmic domain is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein).

b. 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein. There is actually a mixture of normal and abnormal protein present. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.

c. Negative mutations (those that produce inactive protein) are usually NOT dominant.  Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.

d. Significance: What does the existence of a dominant negative mutation imply? It indicates that the gene involved probably codes for a protein that must polymerize in order to act.

    C. How do GPLR's and RTK's Compare? -- See table below for reference for comparison of basic features of  TK or TK linked receptors and G protein linked receptors.  For TK's, see Sadava figs. 15.6 & 15. 10 (15.9) or Becker fig. 14-17 (10-17) for structure and Becker 14-18 (10-18) for mechanism.

Properties of Two main types of Cell Surface Receptors

                                          Type of Receptor 

Property of Receptor G protein Linked Receptor Tyrosine Kinase or TK Linked Receptor (RTK)
Structure Multipass (7X) transmembrane protein# Single pass  transmembrane protein##
What happens on ligand binding Change in conformation Usually aggregation (dimers)##
Receptor activates (binds to) G protein Self (other subunit) or separate TK
Nucleotide role in activation Replace GDP with GTP Add phosphate from ATP to tyr side chains
Activated TK or G protein subunit* binds to: adenyl cyclase or
phospholipase C or
ligand gated channels		 Many dif. proteins, usually protein with SH2 domains**
Can bind a PLC → IP3 etc.
Which 2nd messengers used? cAMP or IP3 etc. IP3, etc. (if any); not cAMP. Often does not use a 2nd messenger.
Receptors for Epinephrine,  ADH, glucagon (most endocrines) Insulin and most GF's such as EGF (many paracrines & autocrines)
Usually modify Enzymes, not TF's Transcription Factors
Affect Transcription? Not usually Often
How contact target? Broadcast Recruitment

# See Becker Fig. 14-4 (10-3). 
## See Becker fig. 14-17 (10-17) or Sadava 15.6 & 15.3.
*Either part, alpha or beta + gamma may be activator/inhibitor; G proteins can also be inhibitory
**SH2 = sarc homology 2 domain

Review problems 6-1 & 6-3

    D. Signaling Pathways all interrelate

1. Different 2nd messengers can influence the same enzyme/pathway. Click here for an example (& problem).

2. Each signaling system can affect the others -- For example, Ca++ levels can affect kinases/phosphatases and phosphorylations can affect Ca++ transport proteins (& therefore Ca++ levels). See an advanced text if you are interested in the details.

3. The same signal (same activated TK receptor) may trigger more than one signaling pathway. For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 14-18 (10-18)).


III. Regulation of the (Normal) Cell cycle  --  see handout 18B .

    A. Overview of Cell Cycle (Sadava fig. 9.3 or Becker fig. 19-1.)

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

2. Role of RTK. Usually required for transition from G-1 to S. 

3. What is wrong with cancer cells? Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at the critical checkpoint --  proceeds too readily from G1 to S.

    B. Mutants (in yeast) indicate the existence of two major control points or checkpoints. See Becker fig. 19-31 (17-30) or Sadava 9.6 (9.4).

1. 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.

2. G1/S (border of G-1 & S) Checkpoint -- also called "start" in yeast or the "restriction point" in mammalian cells

a. 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.

b. This is the major checkpoint/decision point for animal cells in the adult. 

c. Many growth factors (like EGF) act at this point -- signals from neighboring cells are needed to enter S.

3. 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.

4. Additional checkpoints exist.  See texts (especially more advanced ones) if you are interested. 

    C. Basic switch or regulatory protein -- similar in both cases.

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 (17-31) or Sadava fig. 9.4 (in 8th ed.). You need presence of an activator signal (not loss of an inhibitor) to proceed past a checkpoint.  (However, production/activation of the switch protein complex involves a complicated process which can involve multiple inhibitors and/or activators.)

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. 

3. Each kinase phosphorylates a specific set of proteins (See handout 18B)

4. These kinases have 2 parts (See Sadava fig. 9.5 in 8th ed.)

a. CDK = cyclin dependent kinase (called p34, cdc2 etc.). This is the actual catalytic protein. Level of kinase protein itself  remains steady in cycling cells. (Probably degraded if cells stay in G0 = exit the cell cycle.)  Inactive without cyclin; therefore catalytic activity (not amount) of the kinase is dependent on cyclin (& other factors; see below).

b. Cyclin -- builds up, peaks, degraded (in proteasome), repeats (See Becker fig. 19-34 (17-33) or handout 18B for graph). Acts as an activator of the CDK. Cyclin is necessary, but not sufficient, to activate the CDK.

c. Complex of CDK + cyclin forms; inactive; action of right combo of (additional) kinases and phosphatases → active form. (See Becker fig. 19-35 (17-34) if you are curious about the details)

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

5. Effects of kinase activation

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

b. Kinase phosphorylates and thus activates proteins needed to successfully complete next stage of cycle -- start DNA replication, disassemble nuclear membrane, separate chromosomes, etc. (see table on handout 18B)

6. What triggers synthesis of cyclin?

a. G1/S -- Stimulus from outside (usually a Growth Factor) plus internal state of cell. GF uses RTK to activate the TK cascade (see handout 18A).

b. G2/M -- internal state of cell -- is DNA replicated properly, etc.

    D. Regulation of Cyclin

1. Regulation occurs at many levels

a. Cyclin levels are regulated by controlling synthesis of mRNA (transcriptional control) and therefore protein, and by controlling degradation of both mRNA for cyclin and protein itself (post transcriptional control) 

b. Activity of many proteins involved is regulated post translationally by extensive modifications (mostly phosphorylations and dephosphorylations) -- examples include kinases and TF's.

c. Cycle is influenced by external factors (GF's, hormones, contact from other cells, etc.) and by internal factors such as state of the chromosomes, DNA damage etc. Both sets of factors alter passage through the cycle by triggering activations or inactivations of proteins (TF's, kinases, etc.). Regulation of cell cycle integrates both sets of information.

2. Why all these 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 this very carefully controlled. So have multiple "brake" and "accelerator" proteins in this system. Many cancers traced to loss of "brake protein" or over production/activation of "accelerator protein," see below.

To review regulation of the cell cycle, try Problem 15-9.


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

This material is covered in Becker, chap 24 ( Ch17 p. 562 to end), and Sadava sect. 17.4 (Chap 17 pp350-355).

   A. What is wrong with cancer cells?

1. Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at checkpoint --  proceeds too readily from G1 to S.

2. Problem can be at many different steps -- with production of CDK/cyclin complex, with its activation, &/or with its targets. (See handouts 18A & 18B)

3. Two major types of mutations responsible -- as explained previously, these result in either

a. Stuck accelerator (stimulation uncontrolled)

b. Brake failure (inhibition fails)

4. 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

b. Terminology:

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

(2). Over-active version is called an oncogene. (See Becker table 24-1 (17-2) 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 inactive 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 for picture or Becker table 24-2 (17-3) 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 Becker fig. 19-39 (17-40))

(2b). 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 Becker fig. 19-38 (17-36)). 

c. How mutation causes 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.

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

Try problems 15-1, 15-10 & 15-11.

    C. How do Cancer Causing Mutations occur? This section is FYI.

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 Squences. 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 (18.2)) 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. 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.  If you inherit versions of genes giving low repair or high mutability, 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. If you inherit one defective copy of a tumor suppressor gene, nothing happens unless the second copy of the gene gets messed up in a 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 (18.16) 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 (17.18) or Becker fig. 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.)

Try problems 15-3, 15-4, & 15-7.

     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. 14-25 & 14-26.

(3). Normal p53 acts as "brake" on activation of CDK/cyclin start complex. (see handout 18 B, or Becker fig. 19-39 (17-40))

(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 (17-36) & handout 18B.

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.

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: 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.

Try Problems 15-2, 15-5, & 15-6.

   D . 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.) See Sadava 17.19 (18.19).

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

Tamoxifen -- binds to and inhibits an estrogen receptor.

b. Monoclonal antibodies (See Becker box 24B)

Herceptin, Iressa & 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.

By this point, you should be able to do all the problems in Problem Set 15. You do not need to memorize the names of all the proteins and genes involved in the cell cycle. You need to be able to do the problems if you have clean copies (no annotations) of the class handouts in front of you.