Unit 2 - Cell Cycle and Cancer

Once the mutations that conferred resistance to Gleevec were identified, how long did it take for the next drug to be developed?

5 years

Breast Cancer....

Most common form of cancer in US women, but also occurs in men40,000 deaths per yearMost cases are sporadic, but approximately 5% are the result of a mutation in the BRCA1 geneAbout 1/200 women inherit the allele; of these, approximately 90% will develop breast cancer-also link ovarian cancer

What evidence supports this claim?

doctors were able to track a decrease in white blood cell count

List the properties of cancer cells: Tumors

- Uncontrolled proliferation- Ability to metastatize- Are clonal(see pic next page)

What type of gene is APC? What type of gene is B-catenin?

APC is a tumor suppressor gene B-catenin is a proto-oncogene

CML, Philadelphia Chromosome...

Abnormal chromosome produced by a translocation between long arms of chromosome 9 and chromosome 22Linked to chronic myelogenous leukemia (CML)

What would Ms. Meeker recommend Jane do to manage her condition?

Annual sigmoidoscopy and/or colonoscopy

Drawings : Hereditary retinoblastoma

Autosomal dominantIndividuals with the allele, RB have a 90% chance of developing retinoblastoma Usually in both eyes High risk for other cancers especially osteosarcoma and fibrosarcoma(See pic next page)

Drugs

Avastin - prevents angiogenesis does not cure, just prolongs life a bit Herceptin - works on HER2 overexpressing metastatic breast cancer May reduce the risk of relapse by 50%

Rous Sarcoma Virus and the discovery of Oncogenes

Both RNA and DNA viruses can cause cancer. How?Genome of a non-tumor forming retrovirus and Genome of a tumor forming retrovirusCan a vaccine prevent us from developing cancer?(See pic)

Describe the drug that the scientists developed.

Gleevec, a molecule that fits into the ATP-binding pocket and physically prevents ATP from binding there.

Important molecule that regulates p53

Mdm2

Summary of Melissa's story...

Melissa Mills was diagnosed with retinoblastoma in both eyes when she was 11 months old. She talks candidly about growing up with a glass eye, the effect of her cancer on her parents, and how she worried about passing her gene mutations on to her twin girls.

All human body cells express gene C3 . Explain why you would expect gene C3 to be expressed in all body cells

The human body has to go through cell respiration which involves the process of going from ADP to ATP

Describe the three types of cancer genes a) Oncogenes b) Tumor Suppressor Genes c) DNA Repair genes

a)Oncogenes : Normally function to accelerate cell division and growth. When mutated they act like stuck gas pedals. p53 interacts with some oncogenes b) Tumor Suppressor Genes : such as p53, normally act like brakes. Mutations can cause these breaks to fail. c) DNA repair genes : normally function to fix minor damage to DNA when it is replicated. When these genes are mutated , DNA damage can occur and can lead to cancer.

All cells contain gene C4. This gene is not expressed in colon cells. Explain why the C4 gene is not expressed in lung cells

because it is only in red blood cells and not any other genes

Why is it easier to treat a benign tumor than a malignant tumor?

- Benign tumors can be completely removed as long as they are in an operable location - Malignant tumors can be removed but have most likely spread to other parts of the body. Surgical removal is often followed by chemotherapy or radiation which affects healthy cells along with the cancer cells.

Gene Location?

Chromosome 17 and Chromosome 13

Retinoblastoma is...

- Cancer of the retina- RB1 gene at 13q14- Diagnosed between 1-3 years of age- 40% of all cases are due to an autosomal dominant trait60% are sporadic cases

What are the three categories of genes that, when mutated, can lead to tumor formation?

- Tumor suppressor genes - Normally inhibit cell division. If both alleles get are damaged, cell growth is no longer regulated, resulting in excessive proliferation. - Proto-oncogenes- Normally promote growth and division. Only one defective version of an allele of this gene results in excessive proliferation. - Genome stability genes/Caretaker genes- Responds to and repairs DNA damage. Inactivation results in accumulated mutations, some of which will be in oncogenes and tumor suppressor genes.

If the remaining normal APC gene is damaged in one of Jane's cells, how would this affect B-catenin in that cell? How could this affect the growth of that cell?

- Without APC regulation, B-catenin will accumulate in the cell - Cell growth will increase through B-catenin's interaction with TCF to turn on the expression of genes that promote cell division

What are the 3 domains of p53 gene? Explain the functions of each domain?

1) The transaction domain(red) allows p53 to activate other genes after binding to their regulatory regions. The domain recruits RNA polymerase and other enzymes that transcribe RNA. 2) The DNA binding domain (green) is responsible for p53's ability to bind to the regulatory sequences of genes. Most mutations in the p53 protein found in cancers are in this domain. 3)The complexing domain (yellow) is responsible for bringing four individual p53 molecules together.

List characteristics of cancer cells

1. Oilier, less adherent2. Loss of cell cycle control3. Heritable4. Transplantable5. Dedifferentiated6. Lack contact inhibition7. Induce local blood vessel formation (angiogenisis)8. Invasive9. Increased mutation rate10. Can spread (metastatize)

How long did it take researchers to develop Gleevec after the genetic mutation that causes CML was first found?

41 years

What accounts for the difference in development time between the first and second drug?

A combination of advances in molecular biology, structural biology, and DNA sequencing. A combination of advances in molecular biology, structural biology, and DNA sequencing has led to the development of targeted drugs

Why is complexing domain of p53 so important to its function

A single p53 molecule is not really functional. To bind to DNA and activate other genes, it has to form a complex by joining with three other p53 molecules. The domain /region where the four p53 molecules bind to one another is shown in yellow

What are kinases and what do they do?

Kinases are proteins that catalyze the transfer of phosphate groups between molecules. Certain type of kinases regulate and coordinate the cell cycle.

RB1 Gene

Located at 13q14 and encodes for protein pRB pRB regulates the cell cycleIt is present in most cells Activated pRB prevents the cell from moving from G1 to SIf both copies of the RB1 gene are deleted or mutated, the cell divides in an uncontrolled manner(See pic)

Drawings : Sporadic retinoblastoma

Mutations occur in both copies of the RB1 gene at 13q14Tumors form generally in only one eye No increased risk of other cancers (see pic next page)

What can you conclude about gene expression in your patients cells? How is gene expression in your patient different than other 5

My patient has relatively low to medium expression in diseased and normal cells All the other patients had atleast one gene that has high expression in deceased cells compared to normal cells

If someone is predisposed to getting cancer, does that mean that he or she will definitely get cancer some day?

No, just statistically more likely to develop cancer than someone who started with two good alleles of a gene

How does the BCR-ABL protein differ from the normal ABL protein?

Normally, the activity of the ABL kinase is regulated by other molecules. When fused with BCR, the mutant kinase is always active, thus promoting cancer development.

Describe how p53 gene works once activated?

Once p53 is activated, it can bind to a specific sequence of DNA to activate a particular gene. The genes that p53 activates in turn control many functions, including cell cycle and cell death

Cancer causing genes

Oncogenes : Normally stimulate growth, Dominant Suppressor genes : Normally inhibit growth, Recessive

In the cell line that entered mitosis, why do you think that only a fraction of the cells did so instead of 100%?

Only a fraction of cells entered mitosis because even though cancer cells divide rapidly, they don't replicate all the time. The cell needs to undergo some form of gap phases and DNA synthesis to remain alive. Though these phases might not produce normal, well-functioning cells as the DNA is forming many mutations, they are necessary for a cell to split. Thus, only some of the cells are replicating. The other cells are likely synthesizing DNA and doing processes that will keep the cell alive.

What is IVF preimplantation?

Preimplantation genetic testing (PGD) is a screening test that can be performed on embryos created via in vitro fertilization (IVF) to genetically analyze the embryos prior to transfer.

Why did some patients develop resistance?

Resistance occurs because additional genetic mutations change the structure and function of BCR-ABL. The study of additional patients has revealed that two types of resistance mutations can occur

Based on the figure, what role does the p53 protein play in cell division? Why might a cell need to stop dividing?

The role that the p53 protein plays in cell division is that it detects DNA damage and if this damage cannot be fixed, the p53 protein will initiate apoptosis. A cell might need to stop dividing in order to prevent uncontrolled cell growth that might lead to a tumor.

Does Jane have good reason to be worried?

Yes, both her father and brother have been affected by the same type of cancer at an early age. This suggests genetic predisposition.

DNA polymarase

enzyme that forms a new strand of DNA

What does the mutation that converts threonine to isoleucine cause to happen?

A mutation in the DNA that encodes the BCR-ABL protein changes the identity of a single amino acid: the conversion of threonine in the figure above to the larger isoleucine. This change constricts the shape of the ATP-binding pocket so that ATP can still bind but Gleevec no longer fits.

At what point in time were the highest percentage of cells without the p53 gene undergoing mitosis? Compare this with the length of a typical human cell cycle: 24 hours. What could be responsible for this difference in the onset of mitosis?

At about 48 hours, the highest percentage of cells without the p53 gene were undergoing mitosis. The length of a typical human cell cycle is 24 hours. This difference could be caused by the fact that because there are so many cells of the cells that do not have a p53 gene (because they go through uncontrolled cell division), their mitotic division takes much longer.

Predict what might happen at a cellular level and at an individual level if a person has one normal and one mutated allele of the p53 gene. What if they have two mutated alleles of p53?

At the cellular level and individual level, if a person had one normal and one mutated gene they would remain healthy and the cells would be able to create functioning p53 proteins. If one has two mutated alleles, it would be highly susceptible to cancer as there would be no functioning p53 gene to call for the fixing of DNA or apoptosis when necessary.

Describe the mutation that results in the BCR-ABL gene and draw a diagram to illustrate the events that lead to the formation of this gene?

BCR-ABL is a protein produced as the result of a genetic mutation in which parts of chromosomes 9 and 22 are switched. This is called a translocation. The mutated form of chromosome 22 was named the Philadelphia chromosome

Chromosomes involved....

BRCA1 maps to chromosome 17Another autosomal dominant gene, BRCA2 maps to chromosome 13 Together BRCA1 and BRCA2 account for 10-15% of genetic breast cancers Allele frequencies of these genes vary among populationsFrequency of mutations high in Ashkenazi Jewish population(See pic)

The researcher plans to use gene C3 as a positive control gene. Explain why gene C3 is an appropriate positive control

C3 is a good control gene because it adds energy to ADP to turn it into ATP

The researcher plans to use gene C4 as a negative control gene. Explain why gene C4 is an appropriate negative control

C4 is an appropriate negative control gene because it is not produced by colon cells and it is expressed only in red blood cells

What trends do you notice in this graph?

Cells in which both alleles of the p53 gene were disrupted underwent a plethora of mitotic cell division while cells in which both alleles of the p53 gene were not disrupted and cells that contain one normal and one disrupted allele both did not go through too much cell division (both were similar).

What is a reciprocal translocation?

Chromosomal breaks can convert proto-oncogenes to oncogenesIn CMLThe oncogene c-abl is on chromosome 9 at the breakpoint; the bcr gene is on chromosome 22 at the breakpointTranslocation creates a hybrid gene The hybrid protein acts a signal for cell division causing CML(See Pic)

Why was gamma radiation used in this experiment?

Gamma radiation was used in this experiment because it is highly penetrative and therefore it is easier for researchers to study the p53 gene at an in-depth level.

You have been asked to design a drug to inhibit BCR-ABL in an effort to treat CML. Using your knowledge of enzyme catalyzed reactions and the BCR-ABL kinase, propose a drug development strategy to combat CML

Scientists predicted that it might be possible to stop the action of BCR-ABL by blocking ATP from binding. One strategy is to find a molecule that fits into the ATP-binding pocket and physically prevents ATP from binding there.

Abnormal expression of some genes may lead to an increased mutation rate in other genes. From the genes listed in the chart two genes that, if mutated might lead to additional mutations. Support your choices with information from the chart.

The C3 gene since ADP and ATP are form of energy so the cells would need to mutate to lower their energy needs of the C4 gene because this would cause massive cell growth

In the cell line that entered mitosis, why do you think there was a delay before the onset of mitosis after exposure to gamma radiation?

There was a delay before the onset of mitosis after exposure to the radiation because the cells previously undergoing mitosis probably finished replicating. Additionally, the new cells that were just produced were not accustomed to the new gamma radiation exposure. After being exposed to it for a little, severe mutations might have lead new cells that were unable to sustain themselves. This caused a net decrease in the mitotic index. Later the cells that survived began replicating. Only a fraction of them survived as the mitotic index dropped again.

Checkpoint Genes

Tumor suppressor genes Suppress cell divisionAct at either G1/S or G2/M control points Proto-oncogenes Promotes cell division Oncogenes Mutant forms of proto-oncogenes induce or continue uncontrolled cell division

What does it mean to be predisposed to getting cancer?

- A person has inherited one mutant copy of a gene that normally regulates cell growth as a tumor suppressor or a gene that helps maintain genome stability - Only require one more mutation in the normal allele of that gene to cause abnormal cell division

What molecule provides the phosphate for the kinase to transfer?

BCR-ABL

BRCA1 and BRCA2 Are DNA Repair Genes

Expression is highest at the G1/S boundary and S phase of the cell cycleBRCA proteins are activated when DNA is damagedInvolved with repair of double stranded breaks in the DNARegarded as tumor suppressor genes

What is a conformation change mutation?

It's moving back and forth between this inactive and active shape. What these mutations do is force the protein into the active conformation. if you have a mutation now that is making the protein stay in the active form all the time Gleevec's not going to be able to bind. So what I'm showing you on the left is the inactive conformation, and on the right is the mutant form, which creates this active conformation. Now in both forms ATP, the yellow molecule can bind no problem, but now when you try to fit Gleevec in, because of how nicely and tightly it fits into the inactive conformation, it can't fit into the mutant BCR-ABL. (see pic above)

Cyclin Expression cycle

The levels of different cyclins vary considerably across the cell cycle as shown in the diagram. A typical cyclin is present at low levels for most of the cycle, but increases strongly at the stage where it's needed. M cyclin for example, peaks dramatically at the transition from G2 to M phase. G1 cyclins are unusual in that they are needed for much of the cycle(see pic)

How many normal alleles of the p53 gene do cells need to function properly? Use evidence from the figure to support your claim.

To function properly, cells need only one allele of the p53 gene. I inferred this answer because the figure shows the same trend of cell division for cells with one normal allele and one disrupted allele and for cells with two normal alleles.

Did Gleevec work?

Yes

Define Metastasis-Two Types of Genes Involved in Cancer1. Gatekeeper genes2. Caretaker genes

a) Cancer cells break away from their home tissue b) The metastasizing cells become attached to the wall of a blood vessel or lymph vessel. They secrete digestive enzymes onto it. Then they cross the wall at the breach. C) Cancer cells creep or tumble along inside blood vessels, then leave the bloodstream the same way they got in. They start new tumors in tissues,1. Gatekeeper genes Regulate cell growth and passage through cell cycle Example tumor suppressor genes and some oncogenes2. Caretaker genes Help maintain the integrity of the genome Example DNA repair genes

What is transcription factor?

p53 primarily functions as a transcription factor. A protein that regulates the activity of other genes. Part of the reason that p53 can do so many things is that it controls the expression of many other genes.

What kind of inhibitor is the drug?

protein kinase inhibitor

What kind of DNA mutation would result in a single amino acid change?

the conversion of threonine in the figure above to the larger isoleucine

What does it mean if a tumor is classified as "benign"? What does it mean if a tumor is "malignant"?

- Benign tumors do not invade adjacent tissues and do not spread through out the body. They are referred to as 'precancerous' growths and are controlled. - Malignant tumors arise from mutated benign tumors and grow uncontrollably, resulting in cancer. Their cells continue to divide and divide. They metastasize when they invade healthy tissue and spread throughout the bloodstream.

If a cell is stressed, p53 normally functions to shutdown cell division. What activates p53 and what in turn does p53 do?

- p53 normally functions to shutdown cell division when a cell is stressed-for example by DNA damage - When DNA is damaged, p53 activates genes that stop cell growth or even trigger the cell to die - Thus p53 guards against changes to the cells that might lead to tumor formation and cancer. It appears necessary to inactivate p53 to develop many forms of cancer

What happened to the white blood cell counts of the patients represented by the red line, and what are the implications?

After several months on Gleevec the white blood cell counts of some patients started to increase.

reciprocal translocation Linked to what disease?

Chronic Myelogenous Leukemia

The cycle A. B. Control over the cell cycle The cell cycle is controlled at three checkpoints:1. G 1 /S checkpoint-2. G 2 /M checkpoint-3.Late metaphase (spindle) checkpoint

Control over the cell cycleThe cell cycle is controlled at three checkpoints: 1. G1/S checkpoint - 2. G2/M checkpoint - 3. Late metaphase (spindle) checkpoint 4. A Loss-of-function mutation results in reduced or abolished protein function. Gain-of-function mutations, which are much less common, confer an abnormal activity on a protein.

Proto-Oncogenes to Oncogenes• ________________________________ turn cell division on• Mutant forms permanently switch on cell division and are called oncogenes• Many different types of mutations have been identified• Example: ras proto-oncogene receives and transfers signal needed for cell divisionBreast Cancer....Gene

Proto-oncogenes turn cell division on Mutant forms permanently switch on cell division and are called oncogenesMany different types of mutations have been identifiedExample: ras proto-oncogene receives and transfers signal needed for cell division

Which cell line(s) have a properly functioning p53 protein?

The solid bold line has a properly functioning p53 gene because it has two normal alleles of p53. The line with half-shaded also has a properly functioning p53 because it has one mutated allele and one normal allele. Even thought there is one mutated allele the p53 will still properly function.

Cancer drugs

Traditional chemo- and radiation therapy, target all rapidly dividing cells and create serious side effects New drug, Gleevec, targets the specific hybrid protein90% of patients with early stage CML go into remission(See Pic)

What was the next step in solving the problem of Gleevec resistance?

Trying to find compatible mutant drugs

Why might a general ATP-binding inhibitor be a bad idea? Was this a problem with the drug that was developed?

inhibiting a protein kinase that is dysregulated in one organ in a particular disease state may prove harmful to other systems in which that same protein kinase is not dysregulated but instead serves essential functions

How does the BCR-ABL protein promote the development of CML?

Chronic myeloid leukemia (CML) is a cancer of the blood caused by the BCR-ABL protein. CML is characterized by the overproduction of white blood cells

P53 is referred to as a molecule, a protein and a gene. How are these terms related?

Most genes contain the information needed to make functional molecules called proteins. (A few genes produce other molecules that help the cell assemble proteins.) The journey from gene to protein is complex and tightly controlled within each cell.

Role of a p53 gene?

The TP53 gene provides instructions for making a protein called tumor protein p53 (or p53). This protein acts as a tumor suppressor, which means that it regulates cell division by keeping cells from growing and dividing (proliferating) too fast or in an uncontrolled way.

How would you design a follow-up experiment to determine whether other proteins in addition to p53 play a regulatory role in cell division after DNA damage? KEY TERMS allele, cancer, cell cycle, gene knockout, genetic engineering, mitosis, tum

To determine whether other proteins play a regulatory role in cell division after DNA damage, a similar experiment would need to be designed. The same procedure would be used, but the variables would change. Instead of using cells with varying p53 alleles, use cells with varying APC gene alleles. At one level, test cells with an APC gene with two genetically mutated alleles. Then at the other levels have one mutated allele and one normal. At the next, two normal APC alleles.