Cancer Test 2

Esterase D as a marker for LOH in RB patients

-two isoforms of esterase D (A and B). A is larger and travels slower than B. Esterase is located in band 13q14

The Myc family of transcription factors

(Myc, Max, Mxd, and Mxi), which can dimerize in different ways and lead to distinct biological effects of growth, differentiation and and death.

the myc protein, which when expressed in a deregulated fashion, operates as an oncoprotein

(recall the 8, 14; 8, 2 and 8, 22 translocations in Burkitt's lymphoma) 1. Myc's transcription functions require a C-terminal basic helix-loop-helix leucine zipper (bHLHLZ) DNA-protein interaction motif, which is necessary for the interaction with its bHLHZ partner Max and for the sequence-specific binding to a regulatory sequence in the DNA called the E-box.

Knudson's mutation model of retinoblastoma -possibility 1

1) An inherited dominant mutation, the first step to a transformed phenotype 2) A second oncogene is altered in retinal cells leading to cancer

Knudson's mutation model of retinoblastoma -possibility 2

1) An inherited recessive mutation causing the loss of a functional gene suppressing cell growth. 2) A second mutation, this time in the good copy of the gene suppressing cell growth. 3) Without suppression, cell growth is abnormal and cancer results 4) Possibility #2 is the correct one: this led to the designation of the retinoblastoma susceptibility gene as a tumor suppressor gene!

Grb2 and its domains and binding

- Grb2 contains an SH2 domain that binds to phosphorylated tyrosine residues on several receptor tyrosine kinases including the PDGFR and the EGFR. - Grb2 also has two SH3 domains that mediate its binding to the proline rich ligand on SOS, the best example of a Ras GEF. SOS is brought to the membrane by its stable association with Grb2. - GEFs promote the release of GDP from the catalytic pocket of Ras, and the relative abundance of intracellular GTP ensures preferential binding of GTP.

RASSF1A (Ras association domain family 1 isoform A)

- RASSF1A (Ras association domain family 1 isoform A) lacks apparent enzymatic activity but contains a Ras association (RA) domain and is potentially an effector of the Ras oncoprotein. Current evidence supports the hypothesis that it serves as a scaffold for the assembly of multiple tumor suppressor complexes - Originally discovered in the search for a tumor suppressor on chromosome 3p21, subsequent analysis found that epigenetic inactivation of the RASSF1 promoter region by DNA methylation was more widespread in lung cancer than loss of heterozygosity. - Increased expression of DNA methytransferase enzyme going from normal to malignant tissue - Increased promoter methylation comparing normal and more malignant tissue

Proteins that regulate Ras

- Ras activation downstream of receptor protein-tyrosine kinases. A complex of Grb2 and the guanine nucleotide exchange factor SOS binds to a phosphotyrosine-containing sequence in the activated receptor via the Grb2 SH2 domain. This interaction recruits SOS to the plasma membrane, where it can stimulate Ras GDP/GTP exchange. The activated Ras-GTP complex then binds to the Raf, a ser/thr protein kinase. - RAS/GTP's role is to bring RAF to the membrane and Ras may also help to activate Raf by the relief of a raf auto-inhibitory mechanism

The members of the Myc family of oncogenes (N-Myc and L-Myc)

- code for nuclear phosphoproteins that appear to promote cell growth and transformation by regulating the transcription of target genes required for proliferation. 1. Myc has been shown to activate transcription (1.5-2 fold), promote cell growth and prevent cell differentiation when dimerized with Max. Mxd/Max and Max/Max dimers favor differentiation and decreased growth by binding to the E box and repressing transcription.

Propagation of signals to the cell interior

1. (protein kinases, protein phosphatases, lipid kinases, phospholipases, G proteins and nucleotide cyclases) 2. GF=>GFR (tyrosine kinase) => Ras=> kinase cascade (serine/threonine kinases)=> TFs=> cell proliferation -- Growth factor receptors activate G proteins -- Ras proteins are a major point of convergence in receptor tyrosine kinase signaling and are an important component of the cellular machinery necessary to transduce extracellular signals. 3. Levels of myc increase if cancerous, but the structures of ras and src change.

Cell adhesion receptors emit signals that converge with those released by growth factor receptors -functions -FAK

1. 3 Functions: ---Physical link to ECM ---Telling the cell it it is linked ---Facilitating motility 2. ECM-Integrins-Grb-2-SOS-Ras-...-Raf, PI3K and Ral 3. The kinase is focal adhesion kinase (FAK), a non-receptor Tyr kinase 4. Cells undergoing Anoikis are shown in A and cells able to avoid it are shown in B. How might ras play a role in anchorage independence????

Cells make decisions about growth and quiescence during a specific period in G1 phase

1. Almost all cancers have found a way to deregulate the restriction point at G1 2. Cells are responsive to mitogenic GFs and TGF-B during most of G1

Control of pRb function is perturbed in most, if not all human cancers

1. Inactivate Rb by mutation 2. Methylate Rb gene promoter 3. Sequester pRB 4. Overexpress cyclin D or E --High levels of cyclin E lead to extra centrosomes (8) resulting in multipolar spindles and chromosomal chaos/accelerated disease 5. Disruption of INK4 genes

Ras-regulated signaling pathways: -Ras pathway of inositol lipids and the Akt/PKB kinase

1. It turns out that GTP bound Ras can also bind PI3K making it closely associated with the membrane where it can phosphorylate PIP2 to PIP3 attracting proteins with pleckstrin (PH) binding domains. 2. Activation of PI3K by Ras and receptor tyrosine kinases allows it to phosphorylate PIP2 to PIP3 , providing a binding site for AKT/Protein Kinase B 3. PH=pleckstrin domain, able to bind PIP3 4. Activation of AKT/Protein Kinase B after docking on PIP3 by two other kinases PDK1 and 2 5. AKT overexpression and PTEN underexpression is generally not good; cells are much larger -- Ex. Pancreatic cancer

The recessive nature of the cancer cell phenotype -TSG

1. Normal cells have genes which constrain growth, tumor cells have lost these constraints on growth. When a normal cell and tumor cell are fused, the normal cell puts constraints on the tumor cell and the hybrid cell returns to a normal phenotype. 2. Since the wild-type versions of these hypothetical genes antagonize the cancer cell phenotype, the genes were called tumor suppressor genes (TSGs). 3. There were arguments for and against the existence of these so called TSGs!

Promoter methylation represents an important mechanism for inactivating tumor suppressor genes. These are considered epigenetic changes!

-The addition of a methyl group to a cytosine on the 5' side of a guanine; when found in the promoter region shuts down transcription; demethylation can open the gene up for transcription. -In many cancers, the tumor suppressor genes are suppressed by hypermethylation and some oncogenes by hypomethylation. -ex. suppression of RASSF1A by hypermethylation

P21 and P27 actions -and how they influence CDK4/6

1. P21 and p27 don't always work as tumor suppressors since they can also activate CDK 4/6 kinases stimulating cells to progress through G-1 2. Activation of CDK4/6 with D while CDK2 with E is inhibited. Over time, CDK4/6 with D bind up all the p21 and p27, allowing CDK2 with E to no longer be inhibited

Tyrosine phosphorylation controls the location and thereby the actions of many cytoplasmic signaling proteins -src and its domains -v-src

1. The SH1 domain of src contain the catalytic domain 2. The SH2 and SH3 domains of src are involved in substrate recognition and regulation 3. v-src is missing the terminal 7 aa, ends at 526 4. C-src oncogene can result from a mutation resulting at a change in aa 527

Powers of Myc to drive cells from Go thru G1 to S. (Myc ER fusion)

1. The entry of myc/ER fusion protein into the nucleus after binding estrogen is all that is needed to drive a serum starved cell out of G0 and into G1 and S. 2. Other experiments which we will not be discussing show that myc affects 1000s of other genes, many of which have nothing to do with cell cycle progression.

P53

1. The second tumor suppressor gene to be discovered 2. It is located on a 20kb portion on chromosome 17p, band 13.1. The gene consists of 11 exons, the first of which is non-coding and distant from the other 10. 3. The mRNA is 2.2-2.5 kb and is expressed in all cells of the body, especially the spleen and thymus 4. The normal protein is 393 amino acids in length and is a nucleophosphoprotein with a 20 minute half life 5. Abnormal or missing in 50% of human cancers; making it the most common alteration in human cancer. 6. P53 binds to ~300 different gene promoter regions, supporting its potent regulatory role

p53 is discovered to be a tumor suppressor gene

1. To get tumor, you need ras and p53 (deletion or mutant) to get foci 2. Levine's lab made mutations in their cDNA clone and found that many of these mutants transformed cells when transfected together with ras. This suggested that their clone might be the wild type version of p53. -p53 wt did not cause transformation with ras, but Oren's (p53 Val 135) did, as did the p53 KH215 mutant. -Importantly, the transformation seen with Oren's p53 clone plus ras, was suppressed by the addition of wt cDNA in triple transfections suggesting that the wt p53 is a tumor suppressor gene.

LOH events can be used to find tumor suppressor genes -How?

1. Transfection assays, retroviral induction of cancer and gene amplification do not work with tumor suppressor genes despite these techniques leading to the discovery of many oncogenes. 2. Restriction Fragment Length Polymorphism (RFLP) has been an important tool to confirm LOH on various chromosomes 3. Replacing RFLPs in detecting LOH are SNPs (single nucleotide polymorphisms) This technique uses PCR primers complementary for one of the alleles

TGF-B prevents phosphorylation of pRB and thereby blocks cell cycle progression

1. Upon binding TGF-B , type II receptors phosphorylate type I receptors which in turn phosphorylate Smad 3 which then joins Smad 4 and enter the nucleus 2. They increase the transcription of p15 and to a lesser degree p21 3. They also inhibit the transcription of myc

AKT/PKB

-inhibit Bad (inhibit apoptosis) -activate mTOR (stimulate protein synthesis/cell growth) -inhibit GSK-3B (stimulate cell proliferation)

Ways to eliminate wild-type copies of tumor suppressor genes

-mitotic recombination -gene conversion -mutation -chromosomal non-disjunction (lose good copy, or duplicated bad copy) -viral binding of Rb -Mutation upstream of Rb -methylation of promoter (can't transcribe Rb)

Is p53 oncogenic experiment

1. Using the Focus Assay, Land and Weinberg had shown that transformation of primary rat fibroblasts required two oncogenes, either myc plus ras, or E1A plus ras. Oren's laboratory readily showed that his p53 clone plus ras could also transform primary rat embryo cells. 2. This result suggested that p53 was an oncogene. Indeed, another independent publication at the time also came to the same conclusion. However, in contrast to these results, Cathy Finlay in Levine's laboratory could not reproduce this finding with their own cDNA clone; they could only reproduce it when they used Oren's clone.

Myc family dimers

1. When Myc is present, the equilibrium is shifted to Myc/Max dimers, but when Mxd (formerly know as Mad) is present, the equilibrium is shifted to Mxd/Max dimers. In this way, the formation of the heterodimers controls the relative rate of transcription from genes controlled by this family. 2. Myc/max dimers have been shown to increase transcription of cell cycle regulatory genes, such as those encoding Cyclin D, cdk 4, and E2Fs Myc can also dimerize with Miz-1. Myc/Miz-1 dimers repress transcription of p15, p21 and p27.

Further p53 studies (Lane & Crawford, Oren)

1. discovered p53 was complexed with the SV40 T-antigen 2. a cDNA for p53 was isolated. They could isolate full length clones of p53 from the F9 cell (EC cells). 3. The p53 clone had a valine at position 135 while Levine's had an alanine at the same position.

The discovery of p53 (overview)

1. p53 was first described in 1979 as a protein found in SV40-transformed cells, where it was found associated with the T-antigen. 2. Later, the TP53 gene which encodes p53 appeared as a dominant transforming gene in the 3T3 assay - and so was classified as an oncogene. 3. Subsequently it was found that while p53 from some tumor cells was oncogenic, p53 from normal cells positively suppressed tumorigenesis. 4. Thus, in 1989, a better understanding led to its classification as a tumor suppressor gene 5. The response may be to block cell cycle progression or to cause apoptosis depending on the circumstances

A pentanucleotide linked to Rb also shows heterozygosity which can also show loss

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AP-1

AP-1 induces cell cycle progression by leading to transcription of cyclin D gene

SHC

Alternatively, another adaptor protein, SHC, can bind to the cytoplasmic tail of a receptor through its PTB domain resulting in its phosphorylation on tyrosine and subsequently binding Grb2. The exact sequence of binding of adaptors depends on the receptor and cell type.

Pedigree of Hereditary Retinoblastoma (40%); inherited as an autosomal dominant trait with a penetrance approaching 100%

Bilateral cancers, sometimes polyclonal in origin

Hormonal activation of adenylyl cyclase

Binding of hormone promotes the interaction of the receptor with a G protein. The activated G protein α subunit then dissociates from the receptor and stimulates adenylyl cyclase, which catalyzes the conversion of ATP to cAMP.

CDK2/cyclin A use in DNA replication

CDK2/cyclin A is required for the initiation of DNA replication and to support DNA synthesis throughout S phase.

Cyclin, CDK, cell cycle: -cyclin B

Cdk 1-- G2/M

Cyclin, CDK, cell cycle: -cyclin E

Cdk 2--G1/S

Cyclin, CDK, cell cycle: -cyclin A

Cdk 2--S Cdk 1--S/G2

Cyclin, CDK, cell cycle: -cyclin D

Cdk 4--G1 Cdk 6--G1

A signaling pathway reaches the cell surface to the nucleus -early genes

GF binds GFR, signaling the activation of TFs that give rise to immediate early genes which get transcribed and translated into TFs for delayed early gene (depend on TFs often transcribed from immediate early genes)

Are cancer alleles recessive or dominant?

Cross normal cell with cancer cell and see if hybrid cell is tumorigenic

How do cells leave G0 and enter G1?

Rb binds to transcription factors (E2F) on the promoter region of cell cycle genes. The cell cycle genes are turned off until a growth factor signals Rb phosphorylation. Phosphorylation blocks Rb's ability to keep the cell in G1 phase. The cell proceeds to the S phase, during which synthesis of proteins necessary for cell division takes place. However, RB does not work alone, it also recruits histone deacetylases (HDAC)

Cell Cycle Regulation of Rb:

Rb is a target for several pathways that inhibit growth, and may be the means by which growth inhibitory signals maintain cells in G1 (or G0).

Sevenless Gene

Sevenless (sev) is a gene that encodes a receptor tyrosine kinase protein essential to the development of R7 cells in the Drosophila embryonic eye. The R8 photo receptor contains an activator for sevenless called the bride of sevenless (BOSS).

pRb is deployed by the cell cycle clock to serve as a guardian of the restriction point-gate

Since cyclin D is required, this ensures the need for an extracellular signal

pRB function and the controls of differentiation are closely linked

Hypophosphorylated pRB not only inhibits progression through the cell cycle but also favors the differentiation of cells; whereas, hyperphosphorylation favors progression thru the cell cycle and prevents differentiation

P16 (INK4 A): mutations that inactivate the Ink4 Cdk inhibitors are commonly found in human cancer cells

Loss or inactivation of both copies of p16 INK4A 1) Results in CDK4/cyclin D being active 2) Results in inappropriate phosphorylation of Rb 3) E2F is free to cause transcription and HDAC is not available to block it (HDAC removes acetyl groups on histones which allows DNA to be tightly wound and not available for transcription)

Other cancers in patients who have retinoblastoma

Patients with bilateral have 100-fold chance of another mesodermally derived tumor later in life.

Hydrolysis of PIP2

Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol trisphosphate (IP3). Diacylglycerol activates members of the protein kinase C family, and IP3 signals the release of Ca2+ from intracellular stores.

Role of TGF-B in inhibiting cell growth

TGF-B causes increased transcription of p15 in two hours - Mitogens can overcome cell cycle arrest by causing the phosphorylation of p21 and p27 which prevents them from entering the nucleus

E2F transcription factors and pRB

enable pRb to implement growth-versus-quiescence decisions-SKIP except for this figure showing the short window for E2Fs 1-3 have to act. (during late G1 only)

A third way for LOH is simply a mutation in the good copy (Rb mutation)

The RB gene can be mutated causing release of its associated factors. RB mutations have been detected in retinoblastoma and a small fraction of sporadic tumors. RB is sequestered by viral oncoproteins, such as E1A, which prevent it from binding other factors. Phosphorylation (P) of RB by CDK-cyclin complexes during cell-cycle progression disrupts its ability to assemble transcriptional complexes. RB is degraded by a caspase-dependent proteolytic pathway during apoptosis. Except for RB phosphorylation, the other three mechanisms of RB inactivation have been associated with sensitization to apoptosis.

Regulation of Src kinase activity by C-terminal phosphorylation

The normal c-Src proto-oncogene is phosphorylated in vivo at Tyr-527, which down regulates kinase activity. This Tyr has been deleted from the viral Src oncogene protein, which is active as a kinase and phosphorylated at Tyr-416. The significance of Tyr-527 in regulation of Src kinase is demonstrated by mutation of c-src codon 527 to encode Phe instead of Tyr. A c-Src protein with a Phe-527 substitution is like v-Src, activated as a kinase and phosphorylated on Tyr-416.

Regulation of cdk activity by phosphorylation involves both activation and inhibition.

Two phosphorylation sites on the amino terminal end (positions Thr 14 and Thr 15) are inhibitory by preventing ATP binding. Cdc 25, a phosphatase, has to remove those two phosphorylations and CDK-activating kinase (CAK) has to Phosphorylate at Thr 161

The regions of Ras and their purpose

Two regions of Ras, designated switch one and switch two, undergo a conformational change upon GTP hydrolysis. Switch one encompasses residues 30 to 40 and constitutes the majority of the effector loop, which mediates binding to its downstream effectors when Ras is bound to GTP. Switch two, encompassing residues 60 to 76, forms the GTP binding site

Pedigree of the sporadic form of Retinoblastoma (60%)

Unilateral, appears on average by 30 months of age, monoclonal

A fourth way: Chromosomal non-disjunction and loss of heterozygosity

We will also see this Friday, mitotic non-disjunction with chromosomal duplication can account for Rb. RFLP will be the experimental approach used

TGF-β, an anti mitogenic pathway arrests epithelial cells in G1

a. A polypeptide factor that inhibits the proliferation of a variety of types of epithelial cells by arresting cell cycle progression in G1. b. This action is mediated by induction of the transcription of the Cdk inhibitor, p15, which binds to Cdk 4 and 6/cyclin D complexes and weakly thru the transcription of p21. c. Since Cdk4 is inactive, Rb phosphorylation is blocked and the cell cycle is arrested in G1 because Rb remains in an unphosphorylated state (Rb is active and able to recruit HDAC to the DNA and bind E2F preventing it from acting as a TF).

The Wnt--catenin pathway contributes to cell proliferation (and also favors the dedifferentiated state) -- Two distinct roles of B catenin

a. Combine with TF for transcription of myc and cyclin D1 b. connection to cadherin dimer

Gene targets of E2F

a. Cyclin A b. c-myc, a transcription factors for other genes c. Myc/max is a transcription factor for cyclin D d. cdk4 e. Myc/max is also a transcription inhibitor of p15, p16 and p21

A set of proteins interact at the checkpoints to trigger the next events in the cell cycle. Four key elements:

a. Cyclins (D, E, A and B): regulatory proteins that are required to activate cyclin dependent kinases (CDKs) b. CDKs (p34): activate numerous cellular proteins by phosphorylating them (only work if a cyclin is bound) c. The kinases and phosphatases of the CDKs d. The CDK inhibitors: the Ink4 (paper 6) and the Cip/Kip Families

Mechanism of how RB inhibits cell growth and how E7, the oncogene product of papilloma virus, disturbs this delicate balance within the cell

a. E7- an oncogene product of one of the human papilloma viruses b. E2F transcription factors regulate genes required for S-phase and cell-cycle progression c. Rb/E2F complex is also a repressor of other genes

The signal cascade that leads to the transcription of cyclin D and its translation in the presence of growth factors such as EGF

a. Following mitogen stimulation or growth factor signaling, the transcription factor AP-1 is turned on. AP-1 is a transcription factor for cyclin D. b. The levels of cyclin D increase in response to Ras-dependent kinase cascade as well as many other signaling pathways c. Cyclin D moves into nucleus and is able to turn on CDKs 4 and 6.

Cell Cycle Checkpoints

a. G1: DNA damage checkpoint before entering S b. S: DNA damage/ incomplete replication c. G2: DNA damage/ensure completed replication or block entrance to M d. M: anaphase blocked is chromatids are not assembled properly on mitotic spindle **Cell growth is continuous but replication is not

How Raf pathway works:

a. GTP-bound form: Ras interacts with a number of effector proteins, including Raf b. Activation of the RAF protein-serine/threonine kinase, which phosphorylates and activates MAP kinase kinase (also called MEK). c. MAP kinase kinase is a dual-specificity protein kinase (able to phosphorylate both ser/thr and tyrosine residues) that activates MAP kinases (also called ERK 1 and 2) (extracellular signal-regulated kinase). Once activated, ERKs phosphorylate a variety of targets, including other cytoplasmic substrates including kinases and transcription factors such as AP-1

Mechanisms of Rb inactivation

a. Genetic mutation b. Viral inactivation c. Phosphorylation d. Degradation

What Rb looks like in retinoblastomas and in osteosarcomas

a. Large sequences deleted b. According to your text, The normal Rb gene consists of 10 exons and spans 190 kb of DNA. In a more recent publication they mention 27 exons coding, 200 kb of DNA which code for a 105 kD nuclear phosphoprotein

Motifs w/ myc

a. Leucine zipper b. Helix-loop-helic c. Zinc finger d. Homeobox

Rb is a tumor suppressor that controls the cell cycle as well as being able to inhibit apoptosis: key concepts

a. Loss of both alleles of Rb causes retinoblastoma b. Non-phosphorylated Rb prevents proliferation by binding to the TF E2F c. In the normal cell cycle, phosphorylation of Rb by cyclin-cdk is necessary to free E2F allowing the cell to proceed into S phase; Rb is dephosphorylated during mitosis. d. We have seen that some v-oncogenes of DNA viruses can sequester the non-phosphorylated form of Rb (large T, E7 and E1A) preventing it from binding the TF E2F e. Rb can also inhibit apoptosis, independent of its ability to block cell proliferation

Three ways to perturb Rb

a. Mitogenic factors leading to cyclin D and E and the phosphorylation of Rb b. Loss of the Rb by gene mutation etc... c. Oncogenes of DNA virus binding to Rb -- E1A of adenovirus -- E7 of HPV -- Large T of SV-40 3. Although ras leads to both the transcription of cyclin D and its destruction; synthesis is favored

At the onset of mitosis, chromosome condensation is mediated by cyclin B/CDK1 (previously referred to as cyclin B/cdc 2) complexes.

a. Phosphorylation of histone H1 by CDK1/cyclin B modifies chromatin structure through alterations in nucleosome interactions. b. CDK1/cyclin B also contributes to chromosome condensation by phosphorylating and consequently activating casein kinase which is a topoisomerase II activator. c. CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis, which results in nuclear envelope breakdown. d. Additionally, both cyclin A/CDK1 and cyclin B/CDK1 promote microtubule formation from centrosomes.

The APC Gene (Adenomatous Polyposis Coli )

a. The APC gene is a tumor suppressor gene that is located on band 5q21 b. The stromal cells secrete Wnt; Wnt leads to β-catenin release which enters nucleus and associates with Tcf/Let c. The APC gene encodes a 2842 residue protein. Germline mutations can occur anywhere but cluster in the 5' half and introduce stop codons or frame shifts that truncate the protein. Somatic mutations are found in 80% of colorectal cancers and also occur on the 5' side

How these inhibitors work:

a. p27 blocks the ATP binding site on cdk2 b. p16 blocks the binding site for cyclin D on CDK 4 and CDK 6 (cell cycle doesn't begin) c. Rb is not phosphorylated and therefore Rb inhibits progression through G1 by binding E2F and HDAC.

The Jak-STAT pathway

allows signals to be transmitted from the plasma membrane directly to the nucleus 1. Originally discovered in the context of normal cytokine receptors, specifically interferon (IFN) and IL-6. Phosphorylation of STATs induces dimerization and translocation to the nucleus where STAT dimers bind to STAT response elements (SREs) 2. The Stat transcription factors contain SH2 domains that bind to activated receptor tyrosine kinases. 3. pY (tyrosine) needed for dimerization 4. pS (serine) needed for maximal activity

Dual address signaling pathways contribute to normal and neoplastic proliferation

check pics

cyclin D levels/activity in cell cycle

may not always undergo ubiquitination and proteolytic digestion in the way the other cyclins do but it does migrate to the cytoplasm where it is no longer active

The first data in support of cyclins came from studies of amphibian and sea urchin development where cyclin B accumulates in late S and G2 in preparation for mitosis

mhm

Inhibitors of CDKs -Ink4 family

paper 6 last week (p16 (INK4 A), p15 (INK4 B), p18 (INK4 C), p19, (INK4 D) (homolog of p19 in humans is p14; both are also labeled arf and interact with mdm as well): b. The Ink 4 family can specifically target CDKs 4 and 6 1) Cdk4/cyclin D G1 2) Cdk6/cyclin D G1

Familial Adenomatous Polyposis Coli (APC) also known as Familial Adenomatous Polyposis (FAP)

-- The loss of APC function prevents apoptosis and allows b-catenin to accumulate intracellularly and to stimulate cell growth with the consequent development of adenomas. -rapid cell growth increases the possibility for other growth-advantageous genetic events to occur. This causes alterations in the expression of genes, thereby affecting the proliferation, differentiation, migration, and apoptosis of cells. -the polyps can become cancerous. As a result, APC is considered the gatekeeper of colonic neoplasia. Its mutation/inactivation is the initial step in the development of colorectal cancer in patients with FAP. --Patients inherit one mutated APC allele from an affected parent, and adenomas develop as the second normal allele is lost by deletion, mutation or hypermethylation. This completely removes the tumor suppressive function of APC, thus initiating the growth of adenomatous polyps.

Phosphorylation of Rb occurs in two steps:

1) Following growth factor induction of proliferation or the completion of mitotic division, complexes of the cyclin D/CDK4/6 are principal contributors to cell cycle initiation. 2) Cyclin D/CDK4/6 complexes phosphorylate pRb. During early G1, the hypophosphorylated form of pRb with the help of histone deacetylases (HDAC) binds and inactivates the E2F transcription factor. HDACs remove acetyl groups on histones, which reverses the effects acetylation and allows chromatin condensation. The promoter regions of genes are inaccessible to TFs and genes are turned off. 3) Following the first phosphorylations of pRb, HDAC is released, histones becomes acetylated, chromatin is opened up for transcription leading to the transcription of cyclin E. 4) Cyclin E is transcribed and binds to CDK2= hyperphosphorylate Rb causing the release of E2F. E2F transactivates a series of genes required for DNA replication such as cyclin A, thymidylate synthase and dihydrofolate reductase. 5) While Rb appears to be an important target of cyclin D-associated kinases, cyclin E probably targets additional factors necessary for cell cycle progression. However, the sites on Rb that get phosphorylated are only available after cyclin D with CDK 4/6 have phosphorylated Rb.

G-protein-coupled receptors can also drive normal and neoplastic proliferation -G protein actions and subunits

1. G proteins which are stimulatory and activate adenylate cyclase 2. G proteins which are inhibitory and inhibit adenylate cyclase 3. G proteins which activate phospholipase C 4. Most G proteins consist of three subunits, alpha (40kd), beta (37kd) and gamma (8kd). The beta and gamma function as a complex and the alpha subunit has the GTP binding ability

A variety of mitogenic signaling pathways control the phosphorylation state of pRb

1. GF->GFR->Ras->Cyclins D (hypophosphorylation of RB) and E (hyperphosphorylation of Rb)--> Inactivation of Rb-->Act of E2Fs -->S phase entrance

Cell fusion experiments indicate that the cancer phenotype is recessive

1. Heterokaryon 2. Synkaryon 3. Polykaryon

Viral oncoproteins reveal how pRb blocks advance through the cell's cycle

1. Rb is hypophosphorylated in early and mid G1 and then becomes increasing phosphorylated in Late G1-S-G2 2. Immunoprecipitation experiments a. This slide simply proves that E1A from adenovirus can bind to the Rb protein (E7 from HPV and Large T can act in a similar fashion). (an example of convergent evolution aiding viral replication)

SH2 and SH3 groups explain how growth factor receptors activate Ras and acquire signaling specificity.

1. Receptor->Grb2->Sos->Ras 2. Receptor->Shc->Grb2->Sos->Ras

Retinoblastoma tumors and TSGs

1. Retinoblastoma is a rare childhood cancer of the retina with an incidence of 1/20,000. It initiates during development as a result of Rb inactivation in the developing retina. 2. Rb is a "break" on the progression of cells from Go/G1 into S phase 3. Both copies must be lost to cause cancer (chromosome 13)

Levine and Linzer Experiments

1. SV40: a small DNA tumor virus. Causes hamsters to produce antibodies against the viral "tumor antigens." 2. SV40 infected embryonal carcinoma (EC) (F9) cells (derived from a tumor of the germ cell lineage), have a block to viral early gene expression and do not express these tumor antigens. 3. Antisera from hamsters with SV40-induced tumors was collected. This antisera (T) was then used to search for antigens expressed in murine 3T3 fibroblasts or F9 cells that were either SV40 infected or mock infected. The antibodies immunoprecipitated large T antigen from SV40 transformed murine fibroblasts, along with a protein (p53). These proteins were not found using normal sera (N) or in fibroblasts without SV40 infection. 4. When testing the EC F9 cell line, the SV40 tumor serum immunoprecipitated a protein of the same 54 kDa with or without infection

The Ras protein -signaling for compound eye -photoreceptor development

1. Signaling by the Ras/Raf/MAP kinase pathway plays a key role in development of the compound eye of Drosophila. The Drosophila compound eye consists of about 800 individual units, each of which contains eight photoreceptor neurons (R1 through R8) and 12 lens cells. 2. The photoreceptor neurons develop in a fixed order, beginning with the differentiation of R8 (not seen in photo since it is below R7). R8 then induces two neighboring cells to become the R2 and R5 photoreceptors. Next, R2 induces neighboring cells to become R1 and R3, and R5 induces neighboring cells to become R4 and R6. The final step is differentiation of R7, which is induced by interaction with R8

The Ras->Raf-> MAP Kinase Pathway

1. The MAP kinase pathway refers to a cascade of protein kinases that are highly conserved in evolution and play central roles in signal transduction in all eukaryotic cells. 2. The central elements in the pathway are a family of protein-serine/threonine kinases called the MAP kinases (for mitogen-activated protein kinases) that are activated in response to a variety of growth factors and other signaling molecules. 3. In mammals, MAP kinases are ubiquitous regulators of cell growth and differentiation. 4. Placing a PO4 group on a serine (90%) or a threonine (10%) is adding a bulky, highly negatively charged (-2) group to a region of a protein that was only moderately polar is a significant change. This addition will attract positively charged R groups and potentially change the tertiary and quaternary structure of the protein and activate or inhibit function

Inhibitors of CDKs -Cip/Kip

Cip/Kip family, paper 9 this week, (p21, p27, p57): bind to CDKs 1, 2, 4 and 6 (share a broad specificity for binding to and inhibition of most CDK/cyclin complexes (can activate cdk 4 and 6)

Raf and Ras binding -viral Raf oncogene

Raf is able to bind to the effector loop of Ras bound to GTP but not to Ras/GDP. In the viral Raf oncogene protein, the regulatory domain has been deleted and replaced by viral Gag sequences. As a result, the Raf kinase domain is constitutively active, causing cell transformation

Evidence of cytoskeletal changes with the addition of GFs to NIH 3T3 cells

Thanks to integrins linked to actin stress factor for cell motility