C2006/F2402 '09 OUTLINE OF LECTURE #25
Dr. Deborah Mowshowitz, Columbia University, New York, NY.
Last update 05/05/2009 03:03 PM . (A few minor typos fixed after am class.)
I. The Big Question: What is wrong with tumor cells? Usually G1-S switch is defective. Cycle is normal length (not shorter), but cell does not pause normally at the usual decision point -- proceeds too readily from G1 to S. (Additional changes occur as well; see below.) To understand what is wrong, we need to look at the normal cell cycle, and then how it goes wrong in tumor cells.
II. Regulation of the (Normal) Cell cycle -- see handout 25A .
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 multiple things. G1-S switch is defective, which allows cell to multiply; additional mutations allow the cells to metastasize (spread to other locations).
B. Mutants (in yeast) indicate the existence of two major control points or checkpoints. See Becker fig. 19-31 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 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 25A)
4. These kinases have 2 parts (See Sadava fig. 9.5 in 8th ed.)
→ active form. (See Becker fig. 19-35 if you are curious about the details)
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 or handout 25A 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
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 25B)
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 25B).
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.
III. Growth Factors, Growth Control, & Cancer -- Putting it All Together
This material is covered in Becker, chap 24, and Sadava sect. 17.4 (Chap 17 pp 350-355).
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 -- these result in either
a. Stuck accelerator (stimulation uncontrolled)
b. Brake failure (inhibition fails)
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
(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 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 24-3 (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 below & Becker fig. 19-38).
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.
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 (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 (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. 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. 24-9 (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 & handout 25B.
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.
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.) 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.
How is the 'war on cancer' doing? See NYTimes, 4/09: In Long Drive to Cure Cancer, Advances Have Been Elusive.