C2006/F2402 '04 OUTLINE OF LECTURE #13
(c) 2004 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/04/2004 02:11 PM .
Handouts: Need 13A (Cell cycle revisited); 13B (Hormones)
I. Growth Factors, Growth Control, & Cancer -- Putting it All Together. How does normal signaling control the cell cycle, and what goes wrong in cancer cells?
This material is covered in Becker, chap 17 p. 562  to end and in Purves Ch 18, pp 342-346 [Ch 17, pp 386-391].
A. What is wrong with cancer cells? 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.
1. Two major types of mutations responsible -- result in either
a. Stuck accelerator (stimulation uncontrolled)
b. Brake failure (inhibition fails)
2. Types of mutations that cause cancer are used to unravel normal G1/S signaling pathways & vice versa
B. How do GF's & ras fit in? How do they regulate the cycle? See handout 13 or Purves 15.11 or See Becker fig. 17-39 [17-38].
1. Act at G1 to S switch, not at G2 to M switch.
2. GF binds to & activates a receptor, often a TK linked receptor
3. Receptor activates ras pathway
a. MAP kinases. Ras activates first enzyme in series (cascade) of protein kinases generally called MAP kinases.
b. End result = activation of final kinase in cascade = MAP kinase or mitogen-activated protein kinase. See Purves 15.11 or Becker 17-39 [17-38].
TF's are needed for transcription of genes for cyclin (and CDK if cell wasn't cycling).
4. MAPK phorphorylates & activates TF's .
5. Use of cyclin:Needed for formation of CDK/cyclin complex --> active CDK --> entry into S.
6. Overall Result: GF --> receptor --> ras -- > MAP kinase cascade--> TF --> transcription --> synthesize cyclin --> CDK/cyclin complex forms --> CDK activated --> phosphorylation of target proteins --> entry into S
C. Overview of how mutations mess up G1/S switch signaling
1. Mutation can mess up any step: Can interfere with production of CDK/cyclin complex, activation of complex (kinase), or action of kinase (= can have problem with any step in 6 above)
2. Can over-activate an activating (turn on) gene = "stuck accelerator" type of alteration
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.
(1). Normal activator gene is called a proto-oncogene (Purves 18.15 [17.14])
(2). Over-active version is called an oncogene. (See Becker table 17-2 for a list)
c. Oncogene can "over do it" 2 ways
(1). 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). 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 production of permanently active ras.)
3. Can inactive a blocking gene = "brake failure" type of alteration
a. Normal "brake" genes are called tumor suppressors (see Purves 18.17 for picture or Becker table 17-3 for a list)
b. Examples (more details below)
(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. 17-40)
(2). rb -- protein that blocks cell from entering S until GF activates start kinase. When start kinase is activated it phosphorylates rb and removes the block. (see handout and Becker fig. 17-36).
c. Consequences. 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 problems 15-1, 15-9 & 15-11.
D. How do Cancer Causing Mutations occur?
1. Somatic mutations can cause cancer
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.
b. Changes in Regulation. 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.)
c. Changes in Protein Structure. 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 hybrid, constitutively active TK instead of normal TK whose activity is regulated.
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 .
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 Purves 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. HPV makes a protein that inactivates rb protein.
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.
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 Purves 18.16 [17-15]). Example: retinoblastoma, which causes a high risk of eye and ovarian tumors 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 Purves 18.18.
d. What sort of mutation causes cancer? Is cancer caused by a 'lack of function' mutation or 'gain of function' mutation? Usually both.
Try problems 15-3 & 15-4.
E. Examples: Details of how mutant tumor suppressor genes/proteins can cause cancer. Rb & p53
1. Rb & E2F -- see handout 13A & Becker fig. 17-36
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 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).
f. Led to discovery of importance of E2F
2. 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 DNA or other damage.
(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.
(3). Normal p53 acts as "brake" on activation of CDK/cyclin start complex. (see handout or Becker fig. 17-40)
b. Results of p53 failure: When p53 (or other brake protein) is defective, there is no block to growth, and damaged cell keeps on growing --> cells with mutations --> tumors (sometimes).
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. Note: there is an advanced course on this topic -- the molecular biology of cancer -- W4799.
Try Problems 15-2, 15-5, & 15-6.
F. 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 that allow unregulated growth or metastasis. Examples: Gleevec (A review of Gleevec), tamoxifen. Block constitutive kinase and receptors for hormones respectively.
II. Introduction to Hormones
A. Summary of typical hormone roles and examples. See Becker Table 10-3 or Purves 41.1 (38.1) for list of hormones by type of function (Becker) or by source (Purves)
1. Stress response -- cortisol, epinephrine. Regulate heart rate, blood pressure, inflammation, etc.
2. Maintainance of Homeostasis -- insulin, glucagon. Regulate blood glucose/energy supplies and concentrations of substances in general. Maintain more or less constant conditions = homeostasis. (To be discussed next time.)
3. Regulation of episodic or cyclic events -- estrogen, insulin, oxytocin -- regulate lactation, pregnancy, effects of eating, etc.
4. Growth/overall regulation -- growth factors, tropic hormones -- regulate production of other hormones.
B. Overview of Major Glands & Hormones -- see handout 13B for overview. For a complete list see Purves 41.1 (38.1)
1. Hypothalamus (HT) / Pituitary Axis -- More details below or next time. .
a. Structures: Pituitary has 2 parts (anterior pituitary and posterior pituitary) that develop and function separately; how connected to HT. See handout and/or Purves 41.5 [38.5] & 41.7 [38.5].
b. Hormones of HT & Post Pit.
c. Hormones of Ant. Pit & their targets.
2. Adrenal Medulla & Adrenal Cortex. Structure & major hormones. See Purves 41.11 [38.10].
(1). stimulated by ACTH
(2). produces three major types of steroids = corticosteroids. For structures see Purves 41.12 [38.11].
(a). Glucocorticoids. Ex: cortisol -- involved in long term stress response (after epinephrine wears off --more details after nerves)
(b). Mineralocorticoids. Ex: aldosterone -- regulates salt balance (to be discussed when do kidney)
(c). Sex Steroids -- cortex produces low levels of sex hormones (both androgens and estrogens) in both sexes
(1). Stimulated by nerves
(2). Is neural tissue
(3). Secretes transmitters that act as hormones = neuroendocrines. Known as catecholamines (derived from tyrosine) -- structures next time.
(4). Major hormone = epinephrine (adrenaline); also secretes some norepinephrine (noradrenaline). Note that receptors for these hormones/transmitters are known as adrenergic receptors. Role as transmitters to be discussed later.
3. Pancreas -- secretes glucagon and insulin -- function next time.
4. There are other glands/hormones -- the list so far is not exhaustive but covers the major players. See texts for complete lists.
Try Problems 7-1 & 7-3
III. Details of HT/Ant. Pit. Axis
A. Hypothalamic Hormones
1. Inputs: Neuroendocrine cells in HT produce hormones -- in response to 3 inputs -- neuronal, hormonal, & local conditions. (HT has sensors for some variables such as temperature, osmolarity.)
2. Outputs: Neuroendocrine cells in HT are two kinds:
a. Some have bodies in HT and axons/terminals in posterior pituitary
(1). Release hormones (neuroendocrines) from endings (terminals) in post. pit --> blood supply.
(2). Hormones are peptides. Made in cell body, packaged in vesicles, vesicles travel down MT's to end of neurons, hormones released by exocytosis.
(3). Hormones = ADH (vasopressin) and oxytocin. Peptides are very similar in structure (homologous = share common evolutionary origin) but bind to different (G protein linked) receptors --> dif. effects.
(a). ADH. Affects (primarily) water retention; has 2 names because discovered twice from different effects. Details of action to be described when we get to kidney. (Works through IP3 or cAMP.)
(b). Oxytocin. Affects milk ejection, uterine contractions -- works (at least in part) through IP3 to affect Ca++ and therefore contraction
b. Some cells in HT release hormones from HT itself.
(1). Release hormones into portal vessel (connects two capillary beds) that goes direct to anterior pituitary. See Purves 41.7 [38.5] and handout 13B.
(2). Hormones released by HT affect production/release of other hormones by ant. pit.
(3). Affect on release can be stimulatory (RH's such as ACTH-releasing hormone) or inhibitory (IH's such as prolactin release-inhibiting hormone = PIH) For a complete list see Purves 41.2 [table 38.2]
(4). All HT hormones (except PIH = dopamine) are peptides/proteins
B. Hormones of Anterior Pituitary
1. Tropic Hormones (for names of hormones and target cells see handout 13B)
a. Made by ant. pit and influence other endocrine glands
b. Release controlled by hormones from HT
c. Effect on target tissue
(1). Effect is usually release of another hormone
(2). Hormones released by targets are steroids or act like them (thyroxine)
(3). All tropic hormones work through G linked receptors and cAMP.
d. Three major tropic hormone types -- each type named after its target -- see handout 13B & table below.
2. Other Hormones of ant. pit.
a. GH and prolactin -- "pseudo tropic" hormones
(1). Similar in structure to each other (homologous) and use a special type of TK receptor that triggers DAG etc.
(2). Stimulate production of secretions, but not from endocrine glands.
(a). GH stimulates liver (& possibly other tissues) to produce insulin-like growth factors (ILGF 1 & 2); ILGF's from liver released into blood (act as endocrines); ILGF's from other tissues act as paracrines. (GH has other effects as well.)
(b). Prolactin stimulates mammary (exocrine) gland to produce milk. (Need oxytocin to eject the milk.)
b. MSH etc.
(1). All come from cleavage of single peptide precursor (pro-opio-melanocortin or pomC) that is cut up to give ACTH and MSH etc.
(2). Same precursor can be cut up different ways in different tissues and/or species. Note: this is alternative processing of a protein, not an RNA.
(3). Function of these hormones relatively obscure.
3. Summary of Tropic & "Pseudo-Tropic" Hormones of Ant. Pit
Tropic (or Pseudo-Tropic) Hormone(s) Target Organ Hormones/Secretions Made by Target Organ ACTH (adrenal cortex tropic hormone) or adrenocorticotropin Adrenal Cortex Glucocorticoids, Mineralocorticoids & sex steroids Gonadotropins -- LH and FSH Gonads Estrogens, androgens & progesterone TSH (thyroid stimulating hormone) or Thyrotropin Thyroid Thyroxine GH (growth Hormone) or somatotropin Liver (& others) Insulin-Like Growth Factors (ILGF 1 & 2) or somatomedins Prolactin Mammary Gland Milk
Try Problem 7-2, & 7-4, but skip 5 (of 7-4) for now.
Next time: HT/Anterior Pituitary Axis -- Set up & Regulation of overall circuit (HT --> Ant. Pit. --> target)
IV. Summary of Features of Hormones
-- How to Classify/Keep Track of Hormones & Growth Factors
(or Signal Molecules in General). The following is meant as a check list to help
you keep track of the various signal molecules. It is for reference & study
purposes; it will not be discussed in class.
Some of these questions/categories overlap, and you can't answer all the questions for all the hormones, growth factors, etc., but the list helps to organize the information you do have.
1. Type of Action -- Is it paracrine, endocrine (hormone), growth factor, neurotransmitter, etc.? (See handout 10C or Purves 41.1 [38.1])
2. Chemical nature -- Is it a peptide, amino acid or derivative, fatty acid or derivative, or steroid?
3. Where is it made? In what gland or tissue? (HT? pancreas?)
4. Target Cells -- where does it act? (Muscle and liver or just liver?)
5. Mechanism of signal transduction
A. Location/type of receptor on target cells -- Is receptor on surface or intracellular? TK or G protein linked?
B. Type of signal transduction -- Is there a second messenger? Which one? If none, what links receptor to intracellular events?
C. Intracellular mode of action -- Is there a change in enzyme activity? change in transcription? both? change in state of ion channel? = not what happens, but how?
6. What actually gets done? What happens?
A. Biochemically speaking: Which enzymes, proteins or genes are affected (glycogen phosphorylase activated? Cyclin gene transcribed?)
B. Physiological End Result: Another hormone secreted? Glycogen broken down, & Glucose in blood up? Note the "result" may have several steps, and more than one can sometimes be considered "the end."
C. What's the (teleological) point? What overall function is served by the signal molecule's action?
1. One list of possibilities: Homeostasis, response to stress, growth, maintenance of some cycle;
2. An alternative version of the list: Regulation of rates of processes, growth & specialization, Conc. of substances, and response to stress.
3. The 2 lists are really the same = homeostasis (control of rates & concentrations), response to stress, & regulation of growth (unidirectional and cyclic).