Lectures 48-57 Flashcards
(163 cards)
1
Q
How do you tell apart the phases of mitosis?
A
In human somatic cells, the cell cycle has four phases. The S phase is when DNA synthesis occurs. The M phase is when cell division or mitosis occurs. Between these two are two additional “Gap” or “Growth” phases: G1 and G2. These cell cycle phases can be largely distinguished by DNA content. Cells in the G1 phase have a 2N DNA content whereas cells in the G2 phase (after DNA synthesis is complete) have a 4N DNA
2
Q
What is prophase?
A
The centrosomes or microtubule organizing centers are duplicated during S phase. In prophase, the centrosomes move to the poles. In addition, the nuclear membrane breaks down and chromosomes condense. Each pair of sister chromatids are held together by the centromere.
3
Q
What is prometaphase?
A
In prometaphase, the spindle fibers form. These consist of three types. Astral microtubules
4
Q
What is metaphase?
A
In metaphase, there is alignment of chromosomes in the center of the mitotic spindle. Ty[pically, karyotyping is done at this stage
5
Q
What is anaphase?
A
In anaphase, sister chromatids separate and move to opposite poles, as the cell elongates.
6
Q
What is telaphase and cytokinesis?
A
In telophase, the nuclear membrane re—forms, the chromosomes decondense, and the spindle disappears. Cytokineses then occurs, driven by actin filaments, resulting in the separation of
7
Q
What is meiosis?
A
Meiosis is the process of reductive cell division for germ cells. Germ cells have a 1N DNA content and are haploid, whereas somatic cells have a 2N DNA content
8
Q
What happens in Meiosis I?
A
There is a pairing and separation of homologues. The resulting cell divisions result in daughter cells containing one maternal copy or one paternal
9
Q
What happens in Meiosis II?
A
There is a pairing and separation of sister chromatids.
10
Q
When does crossing over occur and what is it?
A
During Meiosis I, chromosomal crossing—over occurs in which segments of homologous chromosomes exchange genetic material. This is a process of recombination that results in limitless genetic diversity. This explains why meiosis does not occur by simple separation of homologues, but rather involves DNA synthesis plus two distinct cell divisions, meiosis I and meiosis II.
11
Q
What is the synaptomeal complex?
A
The synaptonemal complex is highly ordered and consists of 2 lateral elements and a central element which facilitates recombination between the parental and maternal homologues.
12
Q
What are chiasmata?
A
Chiasma represent remnants of the synaptonemeal complex that play an important functions. They physically hold homologues together until segregation occurs, much like centromeres do for sister chromatids.
13
Q
What keeps X and Y chromosomes together?
A
There is a small region of homology between the X and Y chromosomes that allows their pairing. Crossing—over will only occur in this small region. The main function is to allow physically keeping the X and Y chromosomes together until segregation is ready to occur.
14
Q
What is non-disjunction and when does it occur?
A
Non—disjunction happens when homologues fail to separate in Meiosis I. Resulting germ cells lead to abnormal embryos, most of which most die. Some survive resulting in developmental abnormalities such as Down’s syndrome, where there is an extra copy of chromosome 21. One set of homologues does not separate in meiosis I and then meiosis II occurs normally. all gametes have an abnormal # of chromosomes either one too many or one too little. Al;though nondis can occur in meiosis II as well, it is far less likely since this is a more tightly controlled process.
15
Q
What do microfilaments and actin do during mitosis?
A
The centrosomes or microtubule organizing centers are replicated in prophase. In pro metaphase the spindle fibers form. There are 3 types. Astral microtubules position the mitotic spindle, kinectochore microtubules are attached to the chromosomes, and polar microtubules interdigitate with those emanating from the opposite poles. Cytokinesis is driven by actin filaments.
16
Q
What are the two major ways by which genetic reassortment occurs during meiosis?
A
Independent assortment and crossing over.
17
Q
How do you calculate the mitotic index and what is it?
A
The mitotic index is used to calculate the number of cells undergoing cell division, It is calculated as: Mitotic index=number of mitotic cells/total number of cells
18
Q
How does the Ki-67 antigen work?
A
The Ki-67 antigen is expressed in cells undergoing active division. It can be detected using an antibody in immunohistochemistry. It is commonly used in fixed and embedded tissues samples to identify cells that are proliferating. Brown stain.
19
Q
Describe Flow cyt of DNA content.
A
The flow cytometer is a machine that can measure the amount of fluorescence per individual cell.3For measuring DNA content, cells are incubated with a fluorescent dye called propidium iodide. Propidium iodide intercalates into the genomic DNA. A single cell suspension is run through the flow cytometer as shown in the figure. A laser shines a light with a specific wavelength on each individual cell. A detector measures fluorescence at a distinct wavelength. The number of cells with each amount of fluorescence is then quantitated. G1 cells have 1X fluorescence (in some arbitrary units). G2 and M cells having twice as much DNA are at 2X fluorescence and S phase cells are in between, ranging from 1-2X fluorescence The data is displayed as a histogram (see an example below).
20
Q
What are cyclin dependent kinases?
A
Cyclin-dependent kinases consist of a catalytic subunit (Cdk) and a regulatory subunit (Cyclin). Cyclin-dependent kinases, as the name suggests, require a regulatory subunit called a cyclin for activity. These kinases phosphorylate on either serine or threonine that immediately precede a proline residue.
21
Q
Multiple distinct cyclin/cdk complexes exist in human cells. Name the 4 kinds and where they act.
A
1) cyclin D/cdk 4 and cyclin D/cdk 6 during G1.
2) Cyclin E/cdk 2 transition from G1 to S phase
3) Cyclin A/cdk 2 S phase
4) Cyclin A/cdk1 and cyclin B/cdk 1 transition from G2 to M phase.
22
Q
What two things does the binding of cyclin to cdk do?
A
The binding of the cyclin to the Cdk does two things. First, it provides part of the substrate binding site. Hence the same Cdk (for example Cdk2) will have a distinct substrate specificity depending upon whether it is bound by one cyclin or another (in the case of Cdk2, this would be Cyclin E versus Cyclin A). Second, the cyclin induces a conformational change that allows the substrate to access the catalytic site. Note that Cyclin binding is necessary but not sufficient for Cdk activation.
23
Q
What is needed in addition to cyclin binding to activate a cdk?
A
CAK or Cdk Activating Kinase phosphorylates the catalytic subunit of all cyclin-dependent kinases on a conserved threonine residue (threonine 160). In addition to Cyclin binding, this modification is required for full activation of the Cyclin-dependent kinases.
24
Q
How does binding of cyclin and phosphorylation with CAK cause a conformational change in cdk?
A
Prior to Cyclin binding, a part of the Cdk called the T-loop sits in the substrate binding site and prevents substrate binding. The binding of the Cyclin causes the T-loop to shift, partially exposing the substrate binding site. Phosphorylation of the T-loop on threonine 160 by CAK fully moves the T-loop so that the substrate binding site is completely exposed.
25
What inhibits cdm activity?
Phosphorylation by Wee1 at T14 and T15 leaves negative residues immediately next to the ATP binding pocket making it impossible for the negatively charged ATP to bind there. This renders the Cdk inactive.
26
What reverses Wee1's activity?
These phosphates (left by wee1) must be removed by a phosphatase called Cdc25 in order for the Cdk to be fully active. There are three Cdc25C phosphatases in human cells: Cdc25A, Cdc25B, and Cdc25C.
27
How does positive feedback work for Cdk's?
Cyclin-dependent kinases (a lready active) phosphorylate Cdc25 phosphatases and make them more active to remove the inhibitory phosphates. The Cdks also phosphorylate Wee1 and prevent it from adding the inhibitory phosphates. Thus, as cyclin-dependent kinases become active, there is positive feedback that further enhances their activity.
28
What are the two classes of protein inhibitors of CDK's?
The CIP family and the INK4 family.
29
What does the CIP family do?
The CIP family of inhibitors of Cyclin-dependent kinases consist of three proteins: p21, p27, and p57. The CIP family inhibits all Cyclin-dependent kinases. he CIP family binds to the Cyclin-Cdk complex and induces a conformational change that prevents substrate binding.
30
What does the INK family do?
The INK4 family is specific for Cdk4 and Cdk6. The INK4 family consists of p15, p16, p18, and p19. The INK4 family binds only to the Cdk subunit, preventing it from interacting with the Cyclin.
31
What are Cdk's regulated by?
Cyclin binding, various phosphorylations (CAK, Wee1, Cdc25), and two classes of inhibitors (CIP, Ink4)
32
What are the two E3 ubiquitin ligases that are important for cell cycle progression?
The SCF (SKP-Cullin-F box) protein complex, and the The Anaphase Promoting Complex (APC).
33
What does the SCF (SKP-Cullin-F box) protein complex do?
G1-S. Cyclins D and E. The SCF (SKP-Cullin-F box) protein complex serves as the ubiquitin ligases for Cyclins that are important in the G1 to S transition (Cyclin D, Cyclin E). The SKP ligase is always active and it is phosphorylation of the cyclin that causes it to be recognized by the SCF, leading to its degradation.
34
What are the domains of the SCF?
Catalytic subunit=rbx1 or ro50
Scaffold=cul1
adapter= skp1
Variable component; Fbox protein
35
What does the The Anaphase Promoting Complex (APC) do?
M Phase. Cyclin B and Separase. The Anaphase Promoting Complex (APC) is the ubiquitin ligase for the Cyclins that are important for mitotic progression, mainly Cyclin B*. In contrast to SKP, APC does not require Cyclin B to be phosphorylated. Unlike SKP, APC is not always active but becomes activated by the binding of a co-factor either Cdh1 or Cdc20.**
36
What are the subunits of APC?
catalytic=apc11
scaffold= apc2
adaptor= multiple units
variable component= Cdh1 or Cdc20** the binding of this activates APC.
37
Can the G2 and M phases of the cell cycle be distinguished using flow cytometry? If not, what techniques can be used to do so?
Using just a general DNA probe like propidium iodide you can not differentiate between G2 and M using flow cytometry as both have 4N. However, using a more nuanced probe we can separate out which cells are in which faze. The presence of phosphorylated histone 3 on ser10 is a reliable marker to identify cells specifically in M phase. Therefore you can use a bivariate cell cycle kit that probes with both an anti-H3-ser10 antibody and PI to parse out which cells are in soley M versus G2.
38
Entry into S phase is determined by what?
The phosphorylation of pRB.
39
How does the transition into S phase occur?
Cdk phosphorylation disrupts the interaction of pRb with E2F. Growth factor signaling upregulates cyclin D transcription. Cyclin D/cdk4/6 phosphorylation of pRb, release of E2F. E2F then activates low level of target expression (especially cyclin E). Cyclin E/cdk2 phosphorylation of pRb. Robust E2F activation of genes important for S phase.
40
What triggers DNA synthesis?
CDK phosphorylation.
41
How does cdk phosphorylation pre-start?
Formation of the pre-replication complex (pre-RC) occurs during G1 phase (Cdk 6, Cdt1, Orc1, Orc 2-6). Cdk phosphorylation of proteins involved in DNA replication include licensing factors which ensure initiation occurs only once each round, then factors are degraded. Cdk phosphorylation also causes origin firing.
42
What mediates the entry into mitosis?
Entry into mitosis (G2 to M transition) is mediated by Cyclin B/Cdk1 phosphorylation.
43
What does active mitotic cdk-cyclin stimulate?
Nuclear envelope breakdown, chromosome condensation, mitotic spindle formation, and target protein degradation.
44
What is the signaling cascade that begins mitosis?
Cyclin B levels are increased due to transcriptional regulation (loss of a repressor) Cdc25 is phosphorylated in response to the completion of DNA replication. This activates cyclinB/cdk1. Its phosphorylation of substrates results in mitotic entry. **Its targets include Lamins A and C resulting in nuclear membrane breakdown and condensin which leads to chromatin condensation.
45
What is needed for the transition from M to G1 or to exit mitosis?
This exiting of mitosis requires the activation of APC.
46
How is APC activated and what does it do?
APC becomes activated because it is associated with a co-factor, either Cdc20 or Cdh1. APC has two main substrates. Degradation of Separase by APC results in sister chromatid separation. Degradation of Cyclin B results in mitotic exit. Chromosomes decondense and the nuclear membrane re-forms.
47
How does the Cyclin A/cdk2 complex get activated and what does it do?
It is activated by E2F release from rRb. It has two functions. It binds to the polymerase complex to initiate origin firing. it also binds to Orc1, cdk6 and cdt1 to block reinitiation.
48
What is the concept of a checkpoint control?
an ordered series of events dependent on completion of a previous step. Loss of checkpoint control leads to uncontrolled proliferation (cancer).
49
What is the restriction point?
The point at which a cell becomes committed to DNA replication. It reaches the point do to extracellular signaling with growth factors but beyond this point they no longer need it.
50
What mediates the restriction point?
Phophorylation of pRb in response to growth factors.
51
How is the restriction point altered in cancer?
Often the problem with cancer is their entry into the cell cycle continuously with no appropriate stimulus. the checkpoint is lost and cells proliferate in an unregulated way.
52
What happens in the G1 checkpoint?
DNA damage causes up-regulation of the tumor suppressor p53. A kinase cascade results in phosphorylation of both p53 and its negative regulator Mdm2. Mdm2 is a
ubiquitin ligase for p53. Phosphorylation disrupts their binding and p53 levels rise. p53 is a transcription factor. One of its targets is the gene encoding p21, a cyclin-dependent kinase inhibitor. p21 binds to Cdk complexes and prevents phosphorylation of pRb, leading to arrest prior to S phase. p53 can also cause apoptosis.
53
What does Mdm2 do and what happens when it is phosphorylated?
Mdm2 is a negative regulator (ubiquitin ligase) of the p53 tumor suppressor. When it is phosphorylated in response to DNA damage, this disrupts its ability to bind which causes p53 levels to rise.
54
What is a target of p53 activated in the G1 checkpoint?
p21, a cyclin-dependent kinase inhibitor. p21 binds to Cdk complexes and prevents phosphorylation of pRb, leading to arrest prior to S phase. p53 can also cause apoptosis.
55
What happens in the G2 checkpoint?
For the G2 checkpoint, DNA damage triggers a kinase cascade that results in inhibitory phosphorylation of Cdc25. Thus, there is no activation of Cyclin B/Cdk1. There is also a role for p53 as the cyclin-dependent kinase inhibitor p21 can also inhibit Cyclin B/Cdk1.
56
How does unreplicated DNA prevent entry into M phase?
Cdc25 needs to be phosphorylated to be active. This phosphorylation does not occur until DNA synthesis is complete and a full 4N DNA content is achieved.
57
What does the spindle checkpoint do?
Arrests cells in mitosis until all chromosomes are properly aligned in metaphase. The checkpoint is triggered because of a disruption of the spindle and a loss of attachment of microtubules to the kinetochore. This causes a delay in activation of APC.
58
What are benign cells?
Benign cells are generally considered ones that are undergoing uncontrolled proliferation but this does not result in any manifest human disease. In contrast, malignant cells have unlimited cell renewal and result in what is generally considered to be cancer.
59
What are primary cells?
Cells with a finite lifespan that are directly removed from the organism.
60
What are immortalized cells?
cells which have an unlimited life span
61
What are transformed cells?
immortalized cells that have gained certain traits like anchorage independency or failure to stop growing in response to other cell contact.
62
What are tumorigenic cells?
Transformed cells that have the additional property of being able to form tumors in the animal.
63
How is the morphology of cancer cells different?
The morphology of cancer cells is spindle-shaped, often appearing refractile in the plight microscope. This is thought to be due to cytoskeletal changes including actin depolymerization.
64
How does cancer cell metabolism differ from normal cells?
Cancer cell metabolism is altered such that they rely on glycolysis for their ATP production whereas normal cells use the TCA cycle for this purpose. The enhanced glycolytic rate of tumor cells is not necessarily due to being in a low oxygen environment, but rather is a property intrinsic to the cancer cells themselves. This is referred to as the Warburg effect.
65
What is the Warburg effect?
The intrinsic property of cancer cells using glycolosis for ATP production, or having an enhanced glycolytic rate.
66
How can adherence needs differ in cancer cells?
Cells that are normally adherent such as those of epithelial or mesenchymal origin depend on this attachment for their ability to proliferate. Cancer cells become anchorage independent for growth. This property does not apply to non-adherent cells such as those of the blood.
67
What is contact inhibition?
Normal cells stop growing upon contact with neighboring cells. This is called “contact inhibition”. Cancer cells lose contact inhibition and continue to proliferate even when touching adjacent cells.
68
How do cancer cells undergo changes in ploidy?
There can be losses or gains of genetic material. This can occur because of deletions, gene amplification, as well as chromosomal translocations.
69
What do cancer cells need to grow as a tumor?
In order to grow as a tumor, cancer cells also acquire the ability to recruit blood vessels or cause angiogenesis. To enter the bloodstream and travel to distant sites within the organism, tumor cells also become invasive. This ability to seed at distant sites is called metastasis.
70
How come even when you inhibit p53 and pRb function to overcome an M1 event or senescence, you will eventually get an arrest in growth? What is this called?
It was shown that if these cells could be experimentally manipulated to lose their p53 and pRb proteins, they would continue to proliferate. But again after a finite period in culture, they would now die, in a process called an M2 event or crisis. It was shown that these fibroblasts lacked telomerase expression. Restoration of telomerase expression allowed them to overcome crisis and continue to grow indefinitely.
71
What do senescent cells look like?
Senescent cells become large and flat, having what is referred to as a “fried egg” appearance. These cells have large cytoplasm. They also express an acidic form of Beta-galactosidase that can be detected using a colorimetric assay in which the senescent cells stain blue.
72
How does scenesence differ from quiescence?
Senescence is considered to be an irreversible cell cycle arrest. This is in contrast to quiescence. Removal of growth factors form normal cells results in an inhibition of their proliferation, but this can be reversed by restoration of growth factors. Quiescence is thus reversible, while senescence is not.
73
How does telomere shortening induce scenesence?
Telomeres protect chromosome ends from being viewed by the cell as DNA damage. The double- stranded ends of chromosomes contain specialized DNA that can form t-loops. These loops mask these ends from the DNA damage sensing machinery in the cells. Somatic cells lack telomerase expression. With each succeeding cell division, telomeres shorten. When they shorten enough that they no longer can form t-loops, the cell perceives these as DNA damage and activates the p53 pathway leading to senescence.
74
What if p53 activity is blocked in proliferating cells that lack telomerase?
If p53 is deleted, the cells will continue to proliferate and their telomeres will continue to shorten. When the telomeres disappear, the cells begins to lose coding capacity and undergoes crisis.
75
Telomere shortening is not the only way to induce scenesence. Discuss how else it can happen.
Cells can also undergo senescence by activating the p53 pathway by other means besides telomere shortening. Stresses such as sustained DNA damage that can not be repaired or activation of specific oncogene pathways also trigger p53-dependent senescence.
76
How can cancer cells avoid scenesence or crisis independent of reactivation of telomerase?
However, in 10-20% of cancer cells, a telomerase-indpendent mechanism occurs which is called ALT. It is generalyl though that in ALT cells, telomeres are maintined by a process involving homologous recombination of the telomeric DNA.
77
How can we identify whether a cancer cell is using telemorase or ALT to avoid scenesence?
Use of an inhibitor of telomerase (referred to as TERT here) will prevent the maintenance of telomere length in some cancer cells but not others. Those in which inhibitors of telomerase have no effect on telomere length are considered to be ALT cells.
78
What are the two oncotic mechanisms by which RNA tumor viruses act?
First, as in RSV, the viral genome contains an activated form of a cellular gene; that is, the viral genome itself contains the oncogene. Second, the virus integrates in a genomic locus that results in aberrant expression of a cellular gene.
79
What does the Rous sarcoma virus contain?
An activated form of the cellular gene, src. The mutated and activated form of src that is expressed from the viral genome leads to cellular transformation.
80
What is ALV (Avian Leukemia Virus)'s mechanism?
ALV (Avian Leukemia Virus) inserts into the host genome and can lead to aberrant expression of a cellular gene involved in oncogenesis.
81
How does ALV induce oncogenesis?
Integration of ALV occurs upstream of a variety of cellular genes. Most of these integrations have no consequence. However, should ALV insert upstream of a gene such as myc, this leads to its over expression and oncogenesis.
82
If there is a deletion or point mutation in an oncogene's coding sequence how can this be oncogenic?
A hyperactive protein can be made but in normal amounts.
83
A regulatory mutation of an oncogene can lead to oncogenesis by?
The normal protein is made but greatly overproduced.
84
Gene amplification of an oncogene results in?
Numerous copies of the gene so each one is producing a normal amount of functional protein but the net effect is greatly over expressed protein.
85
Chromosome rearrangement can affect protein production from oncogenes in what two ways?
A nearby regulatory sequence can cause normal protein to be overproduced, or fusion to actively transcribed gene products give a hyperactive fusion protein.
86
What happens when ras is mutated in cancer?
Ras sustains mutations at residue 12 (glycine to valine, G12V) or 13 (glycine to valine, G13V) resulting in loss of GTPase activity. These two residues are immediately adjacent to the GTP binding site. This results in an inability of Ras to hydrolyze GTP to GDP. Ras bound to GTP will always be active.
87
What happens when raf is mutated in cancer? In what cancer is this common?
The Raf gene can be mutated at residue 600 from valine to glutamic acid (V600E). This leads to its constitutive activation as a kinase. This Raf mutation is commonly found in melanoma.
88
What are the 3 ways gene amplification can lead to oncogenesis?
First is for gene products that are normally limiting in the cell. Myc, Cyclin D1, and Cdk4 are all examples.
Second is growth factor receptors such as the EGF receptor (EGFR) or its related Her2 (also called erbB2 or Neu). These typically require ligand binding leading to their dimerization and activation. If the localized concentration of these receptors is sufficiently high it is hypothesized that they will cluster, spontaneously interact and self-activate in the absence of ligand.
Third is the creation of an autocrine loop. An example of this is overexpression of Platelet-derived growth factor (PDGF). Normally growth factors come from other cells and interact with the receptor on the cell surface. PDGF gene amplification results in the same cell expressing both the growth factor and the receptor. This is called an autocrine loop.
89
What are the two ways gene rearrangements due to translocations or inversions can lead to oncogeneis?
Chromosomal translocations can arise either due to reciprocal translocation between two different chromosomes or inversion within the same chromosome. These rearrangements can either involve regulatory regions (altering expression of a normal protein) or coding regions (resulting in expression of a novel, fusion protein).
90
What to species are often involved in gene rearrangement involving regulatory regions results in transcriptional over-expression?
Immunoglobulin enhancer or the T cell receptor enhancer, thus resulting typically in B cell or T cell lymphoma.
91
What is the t(8;14) translocation?
This is a reciprocal translocation found in Burkitt’s lymphoma (B-cell lymphoma) that involves the myc gene on chromosome 8 and the immunoglobulin enhancer on chromosome 14.
92
What is the t(14;18) translocation?
This is a reciprocal translocation found in Follicular B-cell lymphoma that involves the bcl-2 gene on chromosome 18 and the immunoglobulin enhancer on chromosome 14. This results in overexpression of Bcl-2 which is an inhibitor of apoptosis (cell death).
93
What does bcl2 do?
It inhibits apoptosis.
94
What is the inv(11) inversion?
This is an intrachromosomal inversion involving chromosome 11. This puts the promoter of the parathyroid hormone gene upstream of the cyclin D1 gene. As the parathyroid hormone promoter is cell-type-specific, it will only be expressed in parathyroid cells. Cyclin D1 was originally cloned as a gene involved in parathyroid adenoma and called PRAD1.
95
What is the t(9:22) translocation?
In chronic myelogenous leukemia (CML), a diagnostic marker is the Philadelphia chromosome involving a reciprocal translocation of the c-abl gene on chromosome 9 and the bcr gene on chromosome 22. This results in the expression of a novel, fusion protein called Bcr-abl. Breakage occurs within exons of the two genes leading to expression of the fusion protein. This results in the Abl kinase being constitutively active.
96
What is a targeted treatment for CML and how does it work?
An inhibitor that is highly selective for the Bcr-abl fusion protein was identified, and is called Gleevec (Generic name is Imatanib). Gleevec has been highly successful as a targeted therapy in CML. Since normal cells do not express the fusion protein, the inhibitor can be highly selective for CML cells.
97
What is the t(15;17) translocation?
In acute promyelocytic leukemia (APL), there is a reciprocal translocation of the PML gene on
chromosome 15 and the retinoic acid receptor (RAR) gene on chromosome 17. This results in the expression of a novel, fusion protein called PML-RAR.
98
What does activated Ras need to enhance cell proliferation in culture?
Although it is expected that activation of certain oncogenes should enhance cell proliferation, often a single oncogene is not sufficient for transformation of cells in culture. Activated Ras is a good example in which the expression of a second oncogene is needed to observe transformation. myc or EA1 can both serve as a co oncogene for activated ras.
99
How can we demonstrate the effectiveness of co oncogenes in mouse models?
Cooperation between two oncogenes can also be shown in vivo using mouse models. In the example, mutant, activated Ras is expressed in the mouse mammary gland and results in some level of tumorigenesis. Overexpression of myc has only minimal tumorigenic effect. However when Myc is overexpressed at the same time as mutant, activated Ras, tumor formation in the mammary gland is quite accelerated.
100
What oncogene is present and activated in RSV?
Src
101
What suggested the presence of tumor suppressor genes?
There are methods in the laboratory that cause the fusion of different cells. Using this approach, normal cells were fused with tumor cells and it was shown that the resulting cell was, in fact, normal. This suggested that the normal phenotype dominates over the tumor phenotype, arguing for the existence of tumor suppressors.
102
What do DNA viruses have and do?
In contrast to RNA tumor viruses, DNA tumor viruses contain novel oncogenes within their genomes that do not have cellular counterparts. Simian virus 40 (SV40) expresses a large tumor antigen, also called large T antigen, that binds to both cellular p53 and pRb and thereby inactivates their function. Adenoviruses express two distinct proteins that accomplish the same thing. The E1A protein binds to pRb and the E1b protein binds to p53. Most notable are oncogenic forms of the human papilloma virus (HPV). The latter is associated with almost all human cervical cancer as well as a subset of head and neck cancers. HPV expresses the E7 protein that inactivates pRb
and the E6 protein that targets p53 for degradation.
103
What is the two hit hypothesis for retinoblastoma?
There are two forms of human retinoblastoma: sporadic and hereditary. The sporadic form typically occurs in a single eye and is not associated with secondary tumors. In contrast, the hereditary form generally involves both eyes (bifocal) and also involves secondary non-ocular tumors. Alfred Knudsen proposed a “two hit” hypothesis proposing that retinoblastoma requires two mutations, most likely at a single locus.
104
What is an important distinction between oncogenes and tumor suppressors?
Activation of an oncogene is a “gain of function” can arise from mutating a single allele. Tumors suppressors when mutated have a “loss of function” and require effects on both alleles.
105
What kind of mutation is typical for the gene encoding p53?
A missense mutation.
106
Where are the majority of p53 mutations or "hot spots" contained?
The majority of mutations are contained within the DNA binding region of p53. R248 and R273 directly contact DNA whereas the remaining “hot spots” play key roles in the structure of p53 to allow these contacts with DNA.
107
How does DNA damage activate p53?
DNA damage leads to phosphorylation and acetylation of p53, resulting in reduced protein degradation. The increased stability is due to inability to interact with Mdm2, a ubiquitin ligase for p53.
108
How does oncogene activation affect p53 expression?
Oncogene activation by proteins such as Myc, Ras, and E1a leads to the upregulation of a protein called ARF. ARF inhibits Mdm2, thereby upregulating p53. As a transcription factor, p53 up regulates target genes such as p21, a cdk inhibitor or PUMA and Noxa, promoters of apoptosis.
109
What is ARF?
ARF is a protein that is unregulated in response to oncogene activation. It inhibits mdm2 therefore up regulating p53
110
What target genes does p53 up regulate to promote apoptosis?
PUMA and Noxa.
111
What does a frameshift mutation in an tumor suppressor gene typically lead to?
Truncated nonfunctional protein.
112
What tumor suppressor is subject to truncation?
The gene encoding the adenomatous polyposis coli protein (APC) is one such example. APC acts by binding to beta-catenin and facilitating its degradation. Frameshift mutations in the APC gene result in the expression of a truncated protein that no longer can bind to beta-catenin.
113
What does APC do?
APC acts by binding to beta-catenin and facilitating its degradation.
114
What does beta-catenin do?
Beta-catenin plays a key role in transcriptional regulation that promotes cell proliferation. The binding of beta-catenin to APC results in beta-catenin being phosphorylated and therefore targeted for degradation by the SCF ubiquitin ligase.
115
The gene encoding the pRb protein is generally subject to what kind of mutation?
Deletion.
116
What is the Loss of Heterozygosity?
when there is missense or frameshift mutation of one allele of a tumor suppressor, the remaining wild-type allele is deleted. This latter effect is referred to loss-of-heterozygosity or LOH.
117
What are the three ways haploinsufficiency can result in loss of a tumor suppressor function?
1. Reduced expression. Mutation of only one allele can result in reduced expression of the tumor suppressor protein. If there is some threshold level of expression that is needed for full tumor suppressor activity, such a reduced level of expression can be sufficient to cause loss of growth control.
2. Dominant negative effects. When one allele is mutated while the other remains wild-type, there is the possibility of a dominant-negative effect. This can occur, for example, when the protein of interest forms a homo-oligomer. The presence of a mutated form of the protein in complex with the wild-type version results in the mutant form inhibiting the function of the wild-type protein. This mechanism is only relevant when there is persistent expression of a mutated protein in the presence of a wild-type protein. This will not be relevant if the altered allele does not express a protein, such as with a deletion.
3. Transcriptional silencing of wild-type allele. When one allele is inactivated by mutation, the remaining allele can be silenced through some epigenetic means. For example, promoter methylation at the wild-type allele can prevent its expression.
118
How can the p53 pathway be affected in cancer?
Inactivation of the p53 pathway occurs by different mechanisms in human cancer. The ARF gene is subject to deletion, missense mutation, as well as promoter methylation. The Mdm2 gene is often subject to amplification and the Mdm2 protein can be found to be over expressed due to other mechanisms. p53 is found to be subject to missense mutation. Finally, the human papilloma virus E6 protein targets p53 for degradation.
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How can the Rb pathway be altered in cancer?
The p16 gene is subject to deletion or missense mutation as well as promoter methylation. The Cyclin D1 gene is commonly subject to amplification as well as being involved in chromosomal translocations. The genes encoding either Cdk4 or Cdk 6are also found amplified and can be subject to missense mutation that prevents the binding of p16. The Rb gene is subject to deletion as well as mutation. In human cervical cancer, human papilloma virus infection leads to expression of the E7 protein.
120
How is the APC pathway affected in cancer?
APC interacts with beta-catenin facilitating its degradation. Loss of APC leads to upregulation of myc and cyclin D1 due to the transcriptional effects of beta-catenin. APC serves as scaffold with axin to bring GSK-3β and beta-catenin together. Mutations in APC cause expression of a truncated
protein that can not bind beta-catenin. Mutations in beta-catenin block its degradation. Axin is also found to be mutated.
121
How can the Erk pathway be altered in cancer?
The Erk pathway involves growth factor signaling via cell-surface receptors. Growth factors can be overexpressed due to gene amplification. The genes encoding the cell-surface receptors can also be amplified as well as subject to missense mutation resulting in a receptor that is constitutively active as a tyrosine kinase. Ras and Raf are subject to missense mutation. Remember ras can be missense mutated at 12 or 23 blocking GTPase activity and leaving it constitutively active. Raf can experience a missense mutation leaving it constitutively active. *melanoma.
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How can the PIP3 pathway be altered in cancer?
The PI3K pathway is also subject to genetic alterations in human cancer Both PI3K and the downstream kinase Akt can be mutated such that they are constitutively active. PTEN is a phosphatase that prevents PI3K signaling and PTEN is often deleted in human tumors.
123
Colorectal cancer is the best documented example of a human cancer in which a sequential series of genetic events have been identified. Describe progression.
Loss of APC, activation of K-Ras (constit active), loss of smad4 and other tumor suppressors, loss of p53, loss of a lot of other shit.
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Why is the INK4 gene a messy complex locus in cancer?
The INK4A gene encodes two proteins, the cyclin-dependent kinase p16 and ARF, the alternate reading frame protein. p16 and ARF share a second and third exon but each has their own first exon and their own promoter.
Due to the splicing of each respective first exon to the shared second exon, the reading frame for p16 and ARF differs. Thus, two completely different proteins are made and each is under control of its own promoter, making their transcriptional regulation completely independent. This complex locus means that one set of mutations that affect exons 2 and 3 will inactivate two pathways at once: the pRb pathway via p16 and the p53 pathway via ARF.
125
What is the selective pressure to inactivate the p53 pathway instead of the Rb pathway in cancer?
Myc and Ras have been shown to lead to an upregulation of ARF. This inhibits Mdm2 and in turn activates p53. Loss of Rb frees E2F. ARF is also a transcriptional target for E2F. Thus, inactivation of the pRb pathway will upregulate p53. These means of signaling to ARF provide the selective pressure to inactivate the p53 pathway.
126
What are the 6 key steps in metastasis?
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#1. Angiogenesis. When the tumor mass reaches 1-2 mm, angiogenesis factors are produced that induce blood vessel formation. These include activators such as acidic FGF, basic FGF, and VEGF. 6 Inhibitors such as thrombospondin are also reduced in expression.
#2. Invasion. Tumor cells become attached to sub-endothelial extracellular matrices via cell surface receptors. This is followed by protease-mediated degradation of the matrix. Migration via chemotaxis using the degradation products or tumor-associated autocrine motility factors also occurs.
#3. Intravasation. Tumor cells invade through vascular endothelial cells and their sub- endothelial basement membranes and enter the vasculature.
#4. Metastasis. Tumor cells must survive shear of blood flow and attack by immune system, then adhere to endothelial cells of the target organ or the exposed sub-endothelial extracellular matrix basement membranes.
#5. Extravasation. Tumor cells extravasate out of the vasculature and into the perivascular stroma. This is the reverse of invasion.
#6. Secondary growth. At these distant sites, there is formation and growth of secondary tumor
metastasis.
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127
What is oncogene addiction?
It had been proposed that during the process of oncogenesis, tumor cells become dependent upon the activated oncogene pathways for their proliferation. This was referred to as oncogene addiction. Studies of targeted-therapies have confirmed that this is indeed the case. Tarceva, a chemical inhibitor of the EGF receptor, results in cell death in tumor cells but only blocks proliferation in normal cells. This was unexpected as the Erk pathway is generally thought to control proliferation rather than survival. This has since been confirmed for a variety of targeted therapies and serves as the basis for effects on cancer cells versus normal cells. The underlying molecular mechanism remains obscure.
128
What is the difference between Necrosis and Apoptosis?
During necrosis, cells and organelles swell and rupture. This leakage induces an inflammatory response. In contrast, during apoptosis, cells shrink and condense. The organelles and membrane retain their integrity. The remnants are then phagocytosed by neighboring cells or macrophages. There is no inflammatory response. Degradation is so rapid that very few dead cells are seen.
129
What are ways to visualize apoptosis?
Morphology. Cells can be stained with a dye such as acridine orange to more readily detect the clear morphological changes that occur.
DNA ladder. There is nuclease activity which cuts genomic DNA to the size that is covered by a nucleosome, in 200 bp multiples.
Flow cytometry and DNA content. Since the genomic DNA is being degraded, apoptotic cells have a hypo diploid DNA content, that is, less than 2N.
TUNEL assay. The breaks in the DNA that occur during apoptosis can be labeled using a bacterial enzyme terminal deoxynucleotidyl transferase. Biotinylated dUTP is used in the reaction and can be readily detected using a streptavidin that has a fluorescent tag.
Annexin V and phosphatidyl serine flip. Phosphatidyl serine is normally localized to the intracellular side of the plasma membrane. During apoptosis, this undergoes a flip exposing it on the cell surface. This is used as the signal for engulfment by macrophages. Annexin V is a fluorescently labeled dye that will bind to phosphatidyl serine and can be used to identify apoptotic cells.
130
Name some ways apoptosis can be triggered.
Apoptosis is activated in T cells by glucocorticoids and antibody binding to the cell surface. In some cells, specialized cell surface receptors bind certain ligands and trigger cell death by this pathway. DNA damage such as ionizing radiation, ultraviolet light as well as genotoxic drugs can cause an apoptotic response. Oncogene activation is also another trigger. In certain cell types, growth factor withdrawal results in apoptosis.
131
What are the two types of caspases and what do they do?
The initiator caspases (caspase-8 and caspase-9) cleave and activate the executioner caspases. The executioner caspases (-2, -3, -6, -7) in turn cleave cellular substrates.
132
What are the two pathways that can signal for apoptosis?
Extrinsic and intrinsic.
133
Describe the extrinsic pathway of apoptosis.
In the extrinsic apoptotic pathway, there are three main steps. The first is ligand-induced receptor trimerization. The second is recruitment of intracellular receptor-associated proteins. The third is initiation of caspase activation. The mechanism by which the initiator caspase, caspase-8, is activated is through induced proximity with the cell surface receptors, bringing together many pro-caspase 8 molecules. Examples of relevant ligand triggers are Fas-Ligand, Tumor Necrosis Factor (TNF), and TRAIL.
134
What is a hallmark indicator of the intrinsic pathway of apoptosis?
The loss of mitochondrial transmembrane potential is a hallmark of intrinsic apoptosis and is diagnostic of the opening of permeability-transition pores.
135
Describe the singaling cascade of intrinsic apoptosis.
upon the reception of an apoptotic stimulus, Cytochrome C is released from the mitochondria and interacts with a cellular protein, APAF-1, and dATP to form a holoenzyme that cleaves procaspase-9.
136
What does cytochrome C interact with to cleave caspase 9?
Cytochrome C is released from the mitochondria and interacts with a cellular protein, APAF-1, and dATP to form a holoenzyme that cleaves procaspase-9.
137
What does cleavage of caspase 9 initiate?
executioner procaspases.
138
The Bcl-2 Family consists of what 3 kinds of proteins?
1. Anti apoptotic Bcl-2 protein (Bcl2, Bcl Xl)
2. Pro apoptotic BH123 protein (bax, bak)
3. Pro apoptotic BH 3 only protein (bad, bim, bid, PUMA, noxa)
Studies in C. elegans (nematodes) were critical for understanding the Bcl-2 family of proteins in humans. Bcl-2 itself has four homology domains (BH1-4) and is anti-apoptotic. Bax and Bak contain three of these homology domains and are pro-apoptotic. There is a large group of BH3- only proteins that regulate apoptosis and are also pro-apoptotic.
139
What do Bax and bak do?
Bax and Bak are normally cytosolic. A death signal causes them to insert and form pores in the mitochondrial membrane which results in the release of cytochrome c.
140
What does Bcl-2 do?
Bcl-2 binds to Bax or Bak and inhibits their activity.
141
What does Bid do?
Bid is a BIH3-only protein that directly activates Bax and Bak. The interaction of Bid with Bax and Bak induces a conformational change allowing Bax and Bak to insert in the mitochondrial membrane.
142
What do PUMA and Noxa do?
PUMA and Noxa are BH3 only proteins that block Bcl-2 inhibition of Bax and Bak.
143
What can p53 transcriptionally up regulate in the intrinsic apoptotic pathway?
The p53 tumor suppressor mediates a cellular response to DNA damage. It acts as a transcription factor. Its targets for transcriptional upregulation that are relevant for apoptosis are Bax, PUMA, and Noxa.
144
Bcl-2 role in cancer?
Bcl-2 was originally identified as an oncogene in B cell lymphoma. Many other examples have been found that showed a clear need for tumor cells to suppress apoptosis.
145
What are the Steps on the way to a fully differentiated cell?
a.Uncommitted precursor cells – cells with more than one possible fate
b. Determination – the process whereby a cell becomes committed to a single
lineage and acquires the competence to undergo terminal differentiation
c. Differentiation – the process whereby a cell acquires cell type-specific
properties (e.g., a skeletal muscle cell expresses muscle-specific proteins that form a functional contractile apparatus)
146
Describe the steps in skeletal myogenesis.
Skeletal myogenesis – a paradigm for cell differentiation
a. Muscle development
- skeletal muscle cells derive from precursors found in the dorsal halves of
somites
b. Events associated with myoblast differentiation
1. exit from the cell cycle
2. expression of muscle-specific genes
3. fusion to form multinucleated myotubes
4. expression of ACh receptors and other proteins of the neuromuscular
junction
147
What do skeletal muscles derive from?
Precursers found in the dorsal halves of somites.
148
What happens in order for a myoblast to differentiate?
1. exit from the cell cycle
2. expression of muscle-specific genes
3. fusion to form multinucleated myotubes
4. expression of ACh receptors and other proteins of the neuromuscular
junction
149
What are the The MyoD family of muscle regulatory factors (MRFs)?
Muscle-specific transcription factors
150
What do MyoD and Myf5 do?
Aid in determination of a myoblast from a somite.
151
What do myogenin and MRF4 do?
Aid in the terminal differentiation of a nyofiber from a myoblast.
152
What is the Muscle creatine Kinase (MCK) gene enhancer?
It has binding sites for MRFs, MEF2 factors and other transcription factors. These txn factors bind directly to DNA and each other to enhance MCK and other muscle specific genes.
153
How are positive feedback loops incorporated into cell differentiation?
Transcriptional feedback - MRFs and MEF-2 autoregulate their own expression and cross-regulate each others’ expression to provide at the
appropriate time: 1) stability to the determined state (MyoD autoregulation); and 2) amplification and maintenance of the differentiation process (MRF and MEF-2 auto- and cross-regulation)
154
How does MyoD induce cell cycle termination after it becomes a myoblast?
upon removal of growth factor, CyclinD leading to decreased cd4 activity and hypOphosphorylated Rb. Additionally, GF removal leads to increased txn activity of myoD. MyoD induces p21 which inhibits cycline E/cdk2, keeping pRb levels really low allowing the myoblasts to exit the cell cycle.
155
What are stem cells?
cells that can self-renew and generate differentiated cell types
156
What are different types of stem cels?
Adult stem cells – responsible for homeostatic maintenance and repair to injury
(e.g., hematopoietic stem cells; skeletal muscle satellite cells)
b. Embryonic stem (ES) cells – cells derived from the inner cell mass of the
blastocyst that have been placed into culture. They can divide indefinitely and
differentiate into all lineages of the three germ layers in vitro and in vivo.
c. Unipotency - the ability to form a single cell type or lineage (e.g., testes stem
cells)
Multipotency – the ability to give rise to multiple cell types (e.g., hematopoietic stem cells)
Pluripotency – the ability to give rise to all cells of the embryo (e.g. ES cells) Totipotency – ability to give rise to all cells of an organism, including embryonic and extraembryonic tissues (e.g., zygotes)
157
Types of nuclear reprogramming?
Somatic cell nuclear transfer (SCNT) – transfer of a somatic nucleus into an
enucleated oocyte
b. Reproductive cloning – generation of a new organism following SCNT (e.g.,
Dolly the sheep) or other means
c. Therapeutic cloning – generation of a blastocyst following SCNT from which
ES cells are isolated and used as a source of differentiated cells for therapy
d. Induced pluripotent (iPS) cells – reprogramming of somatic cells to an ES cell- like phenotype by expression of pluripotency genes (Oct4, Sox2, Nanog, c-myc).
158
Pluripotency genes?
Oct4, Sox2, Nanog, c-myc
159
What are the challenges to using reprogramming as a therapeutic tool?
a. Potential oncogenicity of required gene
b. Vectors – viruses can cause insertional mutations
c. Low efficiency
d. Potential acquisition of genetic changes during the generation of iPS cells
160
In what way are the Oct4/Sox2/Nanog factors of ES cells like the MRFs of the skeletal
muscle lineage?
They are master regulators.
161
Sister chromatids are a result of?
DNA replication in S phase.
162
When does crossing over occur?
Between homologous chromosomes in meiosis 1.
163
When are sister chromatids separated?
In anaphase of Mitosis and Meiosis II
