1. Pick up study guide today - some time in lab for questions

2. Pick up problem set 8 - first 2 questions graded, due start of lab next week.

3. Please let me know if you have a final exam scheduling conflict; we need to reschedule your final ASAP.

4. Reminder on grades - Probable adjustment after final

5. Check your t-test numbers with Jason or myself; be sure your statistics are correct before concluding your unknowns; email OK - lab report due Fri. 12/6 at start of lecture.

6. End of class today - consult with other groups re. were any of your worms dumpy?

Announcements

Using the “t distribution” table

1. The numerator when calculating t should always be a positive value - ie. use absolute value of (mean 1 - mean 2) or just the difference between the two means

2. If your t value is greater than the value in the table (under the p=0.05 column), then there is less than a 5% probability that there is no significant difference. Another way to say this: if t > than table value, there IS a significant difference.

You should determine how small the probability is that your means do not significantly differ: what is the lowest p in which your t value is larger than table value?

In example, t value was greater than table value at p=0.005; therefore, < 0.5% chance there is NO difference.

Review of lecture 362 of 3 main checkpoints in the cell cycle

1. G1/SIs cell the correct size?Is DNA damaged?

2. G2/MIs DNA fully replicated?Is DNA damage repaired?

2.

1.

What proteins regulate arrest vs. proceed?

2 kinds of proteins:

What do these proteins do when complexed together?

Example of target protein that must be modified in order for cell cycle to proceed from G1 to S:

How does p53 arrest cell cycle?

1) kinases - cdk’s 2) Cyclins - complex with cdk’s

Modify (+P) target proteins needed for either replication (S) or mitosis (M)

Rb (retinoblastoma) normally puts brakes on cell cycle; once modified, releases E2F and cell cycle proceeds

Is a transcription factor that activates p21 which inhibits cdk- G1 cyclin; Rb is not +P/ modified

Severe Sunburn example

1) Healthy individual - skin peels:

UV irradiation from sun causes mutation in DNA (generates T dimers and inhibits normal replication); in response to DNA damage, p53 levels increase. p53 will either cause cell cycle to arrest or if too much damage (this case), trigger apoptosis (programmed cell death); dying skin cells = peeling skin.

2) Individual/cells with p53 mutation - why skin cancer?

No ability to trigger apoptosis; therefore no peeling skin. This failure to arrest DNA-damaged cells, or kill them off, will be repeated in subsequent cell cycles permitting other mutations to accumulate, culminating in neoplastic transformation... tumor formation and cancer.

I. Tumor suppressors - normally arrest cell cycle

II. Proto-/Oncogenes - normally promote cell cycle

III. Translocations and Genomic instability

IV. Colon cancer results form series of mutations

V. Carcinogens

Overview of lecture 37

Breast Cancer Tumor Suppressors

• A small proportion of breast cancer is heritable. Two genes are associated with predisposition to developing breast cancer.– BRCA1 on chromosome 17– BRCA2 on chromosome 13

• Normal function of both is in repair of ds DNA breaks.

Tumor suppressors - normally suppress unregulated cell growth: Rb is brake on cell cycle;

p53 also can arrest cell cycle

KEY POINT To promote development of cancer, are mutations in one or both alleles of a tumor-suppressor gene needed?

II. Oncogenes

• Arise from mutation in normal gene called a proto-oncogene; these promote cell division

• Dominant mutation: one copy is sufficient to cause cancer. (different than tumor-suppressors) - when switched on permanently, uncontrolled cell division.

• First link between specific genes and cancer was proposed by Francis Peyton Rous in 1910 (Nobel Prize, 1966): cell-free extracts from chicken tumors injected into healthy chickens caused new tumors. Some “agent” and substance must be transmitting disease; now known that a virus and DNA/gene are responsible.

Rous Sarcoma Virus (RSV)• Discovered by Harold Varmus and Bishop, 1975-76 (Nobel Prize, 1989).

• A transforming retrovirus (TRV): a cancer-causing single-stranded RNA virus that uses reverse transcriptase enzyme to make ssDNA, then ds DNA, which integrates into host DNA.

• Note: not all retroviruses are TRV’s, not all oncogenes caused by viruses.

• 100’s of oncogenes now known.

• RSV captured c-src gene from chickens during infection and it is now in viral genome in slightly modified form. Now, upon infection, the virus’ v-src transforms chicken cells into sarcomas

Southern Blots Probed with viral src Gene Revealed Cellular Origin of Oncogenes

Infected Infected Uninfected chickenchicken #1 chicken #2 (Negative Control)

c-srcProto-oncogeneSURPRISE!

v-src

Origin of Transforming Retroviruses

Capsid protein Reverse Transcriptase Envelope Protein

Mutation creates oncogene

c- onc

v-onc

Converting a proto-oncogene into an oncogene

1. Point mutations - example of ras, next slide

2. Translocations - example of CML/Philadelphia chromosome

3. Overexpression - c-onc may acquire new promoter or enhancer - increase in transcription.

Ras Proto-oncogene• Mutated in 30% of all cancers.

• A “molecular switch” in the signal transduction pathway connecting growth factors to gene expression controlling cell proliferation: GF receptor Ras TF target genes cell division.

• A single amino acid change in Ras protein can cause constant stimulation of the pathway, even in the absence of growth factors.

KEY POINT To promote development of cancer, are mutations in one or both alleles of a proto-oncogene needed?

III. Translocations and genomic instability

c-abl proto-oncogene becomes part of bcr gene and this hybrid bcr/c-abl oncogene is transcriptionally active; cell cycle becomes deregulated - results in CML, chronic myelogenous leukemia. Single white blood cell with translocation event may act as origin.

**-A reciprocal translocation between chr. 9 and 22

Genomic instability

Events resulting in genomic alterations that are characteristic of cancerous cells - gains/losses of chromosomes, rearrangements, amplification/deletion of genetic material-- caused by 1) defects in DNA repair and replication, 2 ) abnormal chromosome segregation, 3) defects in cell-cycle control

Example: 15% of colon cancer cases are HNPCC(hereditary nonpolyposis colon cancer)

-see changes in microsatellite DNA throughout genome, resulting in thousands of mutations

Intense debate on role that genomic instability plays in cancer: cause vs. consequence of malignancy

IV. Cancers Usually Result from a Series of Mutations in a Single Cell

Tumor suppressor Tumor suppressorsoncogene

Normal -> proliferating -> benign -> intermed. -> late -> cancerous -> colon epithelium adenoma adenoma adenoma adenoma cancer

with villi

Tumor Progression: Evolution at the Cellular Level

Benign tumor (polyp in epithelial cells) is confined

by basal lamina; then additional mutation occurs.

Malignant tumor (carcinoma in epithelial cells) grows very fast, becomes invasive, and metastasizes.

Cancer Cells Evade Two “Safety” Mechanisms Built into the Cell Cycle

1. Once p53 is inactivated, cells with DNA damage don’t arrest from G1 and don’t undergo apoptosis.

2. Telomerase enzyme is activated, avoiding the limit to

cell divisions imposed by telomere shortening.

V. Carcinogens

•Chemicals are responsible for more cancers than viruses.

•Most are pro-carcinogens - must be converted metabolically to become active carcinogens; then they bind DNA and cause point mutations

•Historically, first seen in 1700’s - scrotal skin cancer in people who worked as chimney sweeps as children.•Now, radiologists and farmers develop skin cancer; insulation workers develop lung cancer, etc..

•Chemical carcinogens (tobacco smoke and diet) responsible for 50-60% of cancer-related deaths.

-30% of cancer deaths related to smoking (cigarettes)- polycyclic hydrocarbons are converted in cells and cause mutations to DNA

Genetic testing and predicting/treating cancer

Predictive testingDo you want to know if you have a mutation in a tumor-suppressor gene or proto-oncogene? An increased chance of developing cancer, but no clear answer if you will or will not get cancer.

- what if it involves predisposition to a cancer where medical surveillance could detect cancer early?

breast cancer vs. pancreatic cancer???

Testing for treatment/prognosis difference in how you view a small breast tumor depending on whether it has a mutation in p53 or not???

Know limitations and utilities of these tests