cell biology1

Question 1

phenotype of malignant or cancer cells

Answer 1

cells that are: 1. Unresponsive to normal signals for proliferation control. 2. De-differentiated, that is, lack many of the specialized structures and functions of the tissue in which they grow. 3. Invasive, that is capable of outgrowth into neighboring normal tissues to extend the boundaries of the tumor. 4. Metastatic, that is capable of shedding cells that can drift through the circulatory system and proliferate at other sites in the body. 5. Clonal in origin, that is, they are derived from a single cell.

Question 2

benign tumor

Answer 2

made up of cells that are not invasive or metastatic, but like the cancer cells have lost many of the growth controls and specialized functions of normal cells. They are immortalized.

Question 3

Cancer cells

Answer 3

poorly regulated for growth and division, resulting in uncontrolled proliferation in inappropriate locations. For a cell to change from a normal to neoplastic state, changes in cellular heredity must be involved. Early in life, mutagenic events in somatic cells will produce tumors many years later. For example, increased UV light exposure at an early age is associated with an increased incidence of melanoma. One would like to understand what is different about the structure of the genes of cancer cells and what is different in their regulation that can account for the above changes. This information can be used for patient diagnosis, prognosis and therapy. or as well. It is clear that several genes must simultaneously be changed to transform a normal cell to a malignant one. Mutations in both tumor suppressors and oncogenes are needed. The best evidence for this idea has been found in colon cancer. Loss of growth regulation and an increased mutation rate coupled to loss of programmed cell death (apoptosis) can be particularly deadly.

Question 4

inheritability of cancer

Answer 4

In general, cancer is not considered to be an inherited disease, in that, it is not inherited as a single, Mendelian gene. The etiology of cancer is related to the accumulation of somatic mutations produced by environmental factors. As this accumulation takes time, age is a big factor as well. In fact, susceptibility to cancer is inherited. Carcinogenesis is a multi-step process characterized by the accumulation of many somatic, genetic alterations or mutations. It has been estimated by analyzing the DNA sequence of micro-satellite repeats in some colon cancers that a tumor has over 100,000 somatic mutations! Tumor initiation, promotion, conversion and progression are four of these steps. An early event may be a mutation in a DNA repair gene that increases the rate of obtaining further mutations; examples are p53, BRCA1 and BRCA2, which we will discuss later. Susceptibility to cancer can be inherited either in dominant or recessive fashion.

Question 5

Oncogenes

Answer 5

normally stimulate cellular proliferation (analogous to the “gas pedal” of your car), are activated by mutation often in tumor initiation

Question 6

Anti-oncogenes

Answer 6

oncogenes or tumor suppressors, which normally inhibit cellular proliferation (analogous to the “brake pedal” of your car), are inactivated with mutation when tumor initiation occurs.

Question 7

Cytogenetic analysis

Answer 7

Can give clues to the genetic abnormalities in cancer and is used in clinical diagnosis. Such abnormalities include: Translocations and gene deletions may activate oncogenes or inactivate tumor suppressors. For example, chronic myelocytic leukemia (CML) is associated with the Philadelphia chromosome and also see Burkitt lymphoma. Inactivation of tumor suppressors may occur by LOH (loss of heterozygosity), which is associated with many. Some examples are retinoblastoma (RB) and APC gene in FAP (Familial Adenomatous Polyposis). LOH can occur by several different ways, but the end result is the same-loss of a tumor suppressor. “Knudson theory” said that two hits or events were needed to produce retinoblastoma. Aneuploidy correlates with a poor prognosis in many cancers.

Question 8

Examples of cancers that are inherited as autosomal dominant disorders

Answer 8

Familial Adenomatous Polyposis (FAP-APC gene), Familial Retinoblastoma (RB gene), familial Breast and Ovarian Cancer (BRCA1 and BRCA2 genes) and Wilms tumor syndromes.

Question 9

Familial Adenomatous Polyposis (FAP-APC gene)

Answer 9

Incidence of FAP is about 1/10,000, Like RB, FAP is inherited in an autosomal dominant fashion, in which patients that inherit one defective APC gene will be at higher risk (90% will develop colon cancer by age 50) to develop colon cancer. Cancer develops when the wild-type gene is lost by LOH in cells in adenomatous polyps of the colon during the first 20 years. Thus, these benign adenomatous polyps may become malignant by LOH. Like RB, the APC gene was isolated by positional cloning after it was mapped to chromosome 5q by genetic linkage and LOH studies. The molecular information can be used clinically to identify high-risk patients for therapy. The APC gene encodes a cytoplasmic protein that regulates the localization of the Beta-catenin protein. Beta-catenin is kept at the plasma membrane by being bound to E-cadherin in normal cells. The APC protein causes the degradation of any unbound and free Beta-catenin in the cytoplasm. When the APC is lost in FAP patients, Beta-catenin goes to the nucleus to produce transcription of oncogenes like c-myc. Thus, loss of APC tumor suppressor causes an overexpression of the c-myc oncogene, resulting in cancer!

Question 10

Familial Retinoblastoma (RB gene)

Answer 10

In children with the heritable genetic form of retinoblastoma there is a mutation on chromosome 13, called the RB1 gene. The genetic codes found in chromosomes control the way in which cells grow and develop within the body.[8] If a portion of the code is missing or altered (mutation) a cancer may develop. Inherited forms of retinoblastomas are more likely to be bilateral. The development of RB can be explained by the two-hit model. Heritable predisposition to retinoblastoma is caused by germline mutations in RB1 and is transmitted in an autosomal dominant manner.

Question 11

familial Breast and Ovarian Cancer (BRCA1 and BRCA2 genes)

Answer 11

In Breast and ovarian cancers, there are two similar predisposing genes (BRCA1 and BRCA2). About 5% of woman with breast cancers have inherited mutations in the BRCA1 or 2 genes. These inherited cases therefore display LOH and have only mutant BRCA1 or 2 genes. However in the acquired cases, the situation is different from that seen for RB in that somatic mutations in these genes have not been found in tumors. Therefore, it is believed that mutations in other genes may affect BRCA1 and BRCA2 function indirectly. Both BRCA1 and BRCA2 function in DNA repair and their loss may give rise to the many mutations needed for full-blown malignancy.

Question 12

Wilms tumor syndromes

Answer 12

cancer of the kidneys that typically occurs in children, rarely in adults. Mutations of the WT1 gene on chromosome 11p13 are observed in approximately 20% of Wilms’ tumors. At least half of the Wilms’ tumors with mutations in WT1 also carry mutations in CTNNB1, the gene encoding the proto-oncogene beta-catenin. Wilms tumor, aniridia, genitourinary anomalies, and mental retardation, more commonly known by the acronym WAGR, is a syndrome that affects the development of many body systems.

Question 13

Examples of cancers that are inherited as autosomal recessive disorders

Answer 13

Xeroderma pigmentosa (XP genes), Ataxia-telangiectasia (AT gene), Bloom’s syndrome and Fanconi’s congenital aplastic anemia (FA genes).

Question 14

Xeroderma pigmentosa (XP genes)

Answer 14

Xeroderma pigmentosum, which is commonly known as XP, is an inherited condition characterized by an extreme sensitivity to ultraviolet (UV) rays from sunlight. This condition mostly affects the eyes and areas of skin exposed to the sun. Some affected individuals also have problems involving the nervous system. One of the most frequent defects in xeroderma pigmentosum is an autosomal recessive genetic defect in which nucleotide excision repair (NER) enzymes are mutated, leading to a reduction in or elimination of NER.[6] If left unchecked, damage caused by ultraviolet light can cause mutations in individual cell’s DNA. The causes of the neurological abnormalities are poorly understood and are not connected with exposure to ultraviolet light. The most current theories suggest that oxidative DNA damage is generated during normal metabolism in the central nervous system, and that some types of this damage must be repaired by NER. Inherited mutations in at least eight genes have been found to cause xeroderma pigmentosum. More than half of all cases in the United States result from mutations in the XPC, ERCC2, or POLH genes.

Question 15

Ataxia-telangiectasia (AT gene)

Answer 15

a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. Several of these targets, including p53, CHK2 and H2AX are tumor suppressors. The cell cycle has different DNA damage checkpoints, which inhibit the next or maintain the current cell cycle step. There are two main checkpoints, the G1/S and the G2/M, during the cell cycle, which preserve correct progression. ATM plays a role in cell cycle delay after DNA damage, especially after double-strand breaks (DSBs). Ataxia telangiectasia (A-T) (also referred to as Louis–Bar syndrome) is a rare, neurodegenerative, inherited disease causing severe disability. Ataxia refers to poor coordination and telangiectasia to small dilated blood vessels, both of which are hallmarks of the disease.

Question 16

Bloom’s syndrome

Answer 16

characterized by short stature and predisposition to the development of cancer. BS is caused by mutations in the BLM gene leading to mutated DNA helicase protein formation. Cells from a person with Bloom syndrome exhibit a striking genomic instability that includes excessive homologous recombination and hyper mutation. Bloom syndrome is an autosomal recessive disorder, caused by disease-causing mutations in the maternally- and paternally-dervied copies of the gene BLM.

Question 17

Fanconi’s congenital aplastic anemia (FA genes)

Answer 17

FA is the result of a genetic defect in a cluster of proteins responsible for DNA repair. As a result, the majority of FA patients develop cancer, most often acute myelogenous leukemia, and 90% develop bone marrow failure (the inability to produce blood cells) by age 40. About 60–75% of FA patients have congenital defects, commonly short stature, abnormalities of the skin, arms, head, eyes, kidneys, and ears, and developmental disabilities. Around 75% of FA patients have some form of endocrine problem, with varying degrees of severity. There are 15 genes responsible for FA, one of them being the breast-cancer susceptibility gene BRCA2. They are involved in the recognition and repair of damaged DNA; genetic defects leave them unable to repair DNA. The FA core complex of 8 proteins is normally activated when DNA stops replicating because of damage. The core complex adds ubiquitin, a small protein that combines with BRCA2 in another cluster to repair DNA. At the end of the process, ubiquitin is removed.

Question 18

Retinoblastoma Gene (the RB gene)

Answer 18

This protein acts as a tumor suppressor. Rb restricts the cell’s ability to replicate DNA by preventing its progression from the G1 (first gap phase) to S (synthesis phase) phase of the cell division cycle. Additionally, pRB interacts with other proteins to influence cell survival, the self-destruction of cells (apoptosis), and the process by which cells mature to carry out special functions (differentiation). Inherited Retinoblastoma is a relatively rare, pediatric disorder (1/20,000 infants). Cytogenetic analysis of cells from retinoblastomas showed that the region around chromosome 13q14 often had an abnormal structure. Retinoblastoma cells from some patients lack RB completely. Both copies of RB have been deleted as detected by genomic DNA analysis (PCR or Southern hybridization). Some patients have partial deletions or other rearrangements of RB. In cases of “inherited” retinoblastoma (i.e. when there was a parent and other family members who also had the disease), the DNA from normal tissue of the patient or from other unaffected family members often shows a defect in the retinoblastoma gene, but has one normal copy of the gene per cell. In these patients it appears that normal, nonmalignant retinal cells, are heterozygous for the retinoblastoma gene, but the tumor cells have descended as a clone from a single cell that has acquired homozygosity for the retinoblastoma susceptibility gene. This is the hallmark of a antioncogene or tumor suppressor gene. The normal, non-malignant Rb protein is hyperphosphorylated in rapidly proliferating cells at S or G2 of the cell cycle, but is hypophosphorylated in non-proliferating cells in G0 of G1 of the cell cycle. The hypophosphorylated form of the RB protein normally functions to repress the entry of cells into the S phase of the cell division cycle. When RB becomes hyperphosphorylated, it no longer inhibits this transition and the cells begin a cell division cycle. Thus, when there is no RB protein or it is all nonfunctional, cells cannot down regulate their cell division and grow out of control. Phosphorylation by CDKs (cyclin-dependent protein kinases) inactivates the RB protein, thereby allowing the cell to proceed from G1 to the S phase of the cell cycle. The RB protein is a target for many animal tumor viruses, for example, SV40 and HPV (human papilloma virus). These viruses drive a quiescent cell into the S phase of the cell cycle and to proliferate by producing a viral protein(s), SV40 T antigen (T stands for transforming) or HPV E7 protein, that binds to and inactivate the RB protein.

Question 19

HeLa cells

Answer 19

HeLa cells were isolated from a cervical carcinoma and have been growing in culture for over 60 years. These cells express HPV E7 and E6 protein (E6 inhibits p53, another important tumor suppressor). If E7 and E6 expression is blocked, the cells return to normal phenotype. This bodes well for therapy as affecting just two proteins can have a drastic effect.

Question 20

inheritance pattern of retinoblastoma

Answer 20

What is inherited in a dominant fashion is the susceptibility to retinoblastoma. People who are heterozygotes for retinoblastoma have only one normal RB gene in each cell of their body including the cells of the retina. These cells will regulate their proliferation normally and will be non-malignant. However, loss of the single, normal RB gene by any number of events will produce a tumor. Thus if one cell among the millions of retinal cells has no RB protein, it will lose the ability to regulate its proliferation, grow out of control, thereby generating a clone of cells, which will become a malignant tumor. Thus people who are heterozygotes for the RB gene are likely to develop the disease and will pass on the defective gene to 1/2 of their children, so it appears to be autosomal dominant in its inheritance. In reality, a cell must have a homozygous RB mutation in order to become malignant, because both RB genes in that cell must be inactivated, if it is to grow out of control. Sporadic cases of retinoblastoma (i.e. cases that occur without prior family history) are attributable to two independent events occurring in a retinal cell. In other words, both RB genes must be inactivated as hypothesized by Knudson. Usually sporadic cases have unilateral retinoblastoma because the probability of two events is low and is unlikely to occur in a cell of both retinas. Bilateral retinoblastoma occurs in inherited cases, because only one copy of RB needs to be inactivated. Persons who survive inherited retinoblastoma have an increased risk for developing a second neoplasm, which is typically mesenchymal in origin, for example, osteosarcoma. Cells of these tumors are also defective in RB function. Cells derived from a high frequency of small cell lung tumors and from some breast tumors carry a defect in the RB gene. Thus while normal RB function is required to suppress a specialized tumor of the eye, it may also suppress tumors in other cell types. In Rb -/- knockout mice, loss of RB results in pituitary tumors with 100% penetrance. It is still not clear why retinal cells are mainly affected in the inherited cases in humans.

Question 21

the importance of membranes

Answer 21

Life requires membranes. Membranes are composed of lipids, carbohydrates, and proteins and function as physical barriers that define boundaries. Membranes spontaneously form sealed structures. Proteins that span the membrane control the movement of molecules between the inside and outside of the structure (cell or organelle). The plasma membrane defines the boundary of the cell and membrane proteins sense the extracellular environment. Organelles are membrane-bound compartments that have specific structures and functions. Each membrane type has a unique complement of proteins and lipids. Lipids, which form the primary structure of the membrane, often have carbohydrates attached on the extracellular surface. Proteins embedded in the membrane also often have carbohydrates attached to the extracellular domains of the protein.

Question 22

Lipid Bilayer

Answer 22

All cell membranes are lipid bilayers (~ 5 nm in thickness) with proteins embedded in or associated with the bilayer. Most water soluble substances cannot pass through the bilayer unless a protein enables the passage of the substance. Proteins spanning the lipid bilayer mediate many of the functions of the membrane (~30% of the all proteins encoded in the genome are membrane or membrane-associated proteins). Some signaling pathways depend on cleavage or phosphorylation of membrane lipids. Lipid bilayers are dynamic and fluid structures; membrane fluidity depends on composition and temperature. The typical lipid molecule exchanges places with its neighbors in a bilayer 107 times/second and diffuses several mm/second at 37°C within a lipid bilayer leaflet. Phospholipids do not spontaneously flip-flop in membranes. For specific functions, an ATP driven Flippase catalyzes flip-flop.

Question 23

Carbohydrates role with cell membrane

Answer 23

Carbohydrates on membrane proteins and lipids are exceedingly important for development, immunological responses, binding of viruses and toxins, and for proper protein folding. Carbohydrates are on the extracellular side of the plasma membrane.

Question 24

Molecules Forming the Lipid Bilayer

Answer 24

There are three classes of lipids in a membrane and all 3 classes are amphipathic (contain hydrophilic and hydrophobic domains). All are synthesized in the endoplasmic reticulum (ER). The three classes are phospholipids, sphingolipids, and cholesterol.

Question 25

phospholipids

Answer 25

The most common phospholipids are phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). All the lipid molecules shown are derived from glycerol except for sphingomyelin, which is derived from sphingosine.

Question 26

sphingolipids

Answer 26

a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine.

Question 27

cholesterol

Answer 27

Cholesterol has a polar hydroxyl group, a rigid steroid ring group, and a hydrocarbon tail. Cholesterol is intercalated among membrane phospholipids. The interaction of the steroid ring with the hydrophobic tail of other phospholipids tends to immobilize the lipid and decrease fluidity. Lipids are forced to be straightened by cholesterol. The thickness of a membrane depends on the amount of cholesterol. Intracellular membranes have less cholesterol than the plasma membranes and are thinner than the plasma membranes. The mole percentage of cholesterol roughly doubles from the ER (7%) to the Golgi (13%) and again from the Golgi to the plasma membrane (26%). Cholesterol is thought to be distributed equally in the two leaflets/monolayers. Cholesterol is extremely important for membranes and its abundance is closely regulated in the body.

Question 28

negatively charged phospholipids

Answer 28

Negatively charged phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) are more abundant on the internal surface.

Question 29

positively charged phospholipids

Answer 29

PC, sphingomyelin, and glycolipids are more abundant on the external surface.

Question 30

phosphatidylserine (PS)

Answer 30

Phosphatidylserine(s) are actively held facing the cytosolic (inner) side of the cell membrane by the enzyme flippase. This is in contrast to normal behavior of phospholipids in the cell membrane which can freely flip their heads between the two faces of the membrane they comprise. However, when a cell undergoes apoptosis phosphatidylserine is no longer restricted to the cytosolic domain by flippase. When the phosphatidylserines naturally flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf the cells

Question 31

phosphatidylinositol (PI)

Answer 31

Phosphatidylinositol is classified as a glycerophospholipid that contains a glycerol backbone, two non-polar fatty acid tails, a phosphate group substituted with an inositol polar head group.

Question 32

phosphatidylethanolamine (PE)

Answer 32

It can mainly be found in the inner (cytoplasmic) leaflet of the lipid bilayer. As a lecithin, PE consists of a combination of glycerol esterified with two fatty acids and phosphoric acid. Whereas the phosphate group is combined with choline in phosphatidylcholine, it is combined with the ethanolamine in PE.

Question 33

phosphatidylcholine (PC)

Answer 33

are a class of phospholipids that incorporate choline as a headgroup. Phosphatidylcholine is a major constituent of cell membranes and pulmonary surfactant, and is more commonly found in the exoplasmic or outer leaflet of a cell membrane. It is thought to be transported between membranes within the cell by phosphatidylcholine transfer protein (PCTP).

Question 34

Regulation of cholesterol

Answer 34

We get cholesterol from (1) ingestion and uptake and (2) synthesis by the liver. Uptake depends on the low density lipoprotein receptor (LDLR). The fundamental concept is that there is negative feedback for cholesterol production; if you get enough in the diet, you decrease synthesis and vice versa. Elegant mechanisms regulate cholesterol synthesis. Synthesis depends on approximately 30 enzymes. The first and rate-limiting enzyme in this pathway is HMGCoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase); statins, used to lower cholesterol, block this step. Both uptake and synthesis are regulated by the sterol regulatory element binding protein (SREBP) Steps: 1. When cholesterol levels are low SCAP-SREBP complex dissociates from Insig. 2. SCAP escorts SREBP to the Golgi by vesicular transport. 3. The bHLH transcription factor is released from SREBP by two step proteolysis- RIP- Regulated Intramembrane Proteolysis 4. S1P is luminal, S2P is within the membrane – cleavage by both is required for activation 5. Nuclear bHLH SREBP moves to the nucleus, binds to DNA promoters, and activates many genes to produce more LDLR to bring cholesterol into the cell and to increase all the enzymes involved in cellular synthesis of cholesterol.

Question 35

HMGCoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase)

Answer 35

the rate-controlling enzyme of the mevalonate pathway, the metabolic pathway that produces cholesterol and other isoprenoids. this enzyme is suppressed by cholesterol derived from the internalization and degradation of low density lipoprotein (LDL) via the LDL receptor as well as oxidized species of cholesterol. Competitive inhibitors of the reductase induce the expression of LDL receptors in the liver, which in turn increases the catabolism of plasma LDL and lowers the plasma concentration of cholesterol, an important determinant of atherosclerosis. This enzyme is thus the target of the widely available cholesterol-lowering drugs known collectively as the statins. HMG-CoA reductase is anchored in the membrane of the endoplasmic reticulum, and was long regarded as having seven transmembrane domains, with the active site located in a long carboxyl terminal domain in the cytosol. HMG-CoA reductase is active when blood glucose is high. The basic functions of insulin and glucagon are to maintain glucose homeostasis. Thus, in controlling blood sugar levels, they indirectly affect the activity of HMG-CoA reductase, but a decrease in activity of the enzyme is caused by an AMP-activated protein kinase, which responds to an increase in AMP concentration, and also to leptin. HMG-CoA reductase is phosphorylated and inactivated by an AMP-activated protein kinase, which also phosphorylates and inactivates acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis. Thus, both pathways utilizing acetyl-CoA for lipid synthesis are inactivated when energy charge is low in the cell, and concentrations of AMP rise. There has been a great deal of research on the identity of upstream kinases that phosphorylate and activate the AMP-activated protein kinase. Rising levels of sterols increase the susceptibility of the reductase enzyme to ER-associated degradation (ERAD) and proteolysis.

Question 36

Transcription of the HMG-CoA reductase

Answer 36

Transcription of the reductase gene is enhanced by the sterol regulatory element binding protein (SREBP). This protein binds to the sterol regulatory element (SRE), located on the 5’ end of the reductase gene. When SREBP is inactive, it is bound to the ER or nuclear membrane with another protein called SREBP cleavage-activating protein (SCAP). When cholesterol levels fall, SREBP is released from the membrane by proteolysis and migrates to the nucleus, where it binds to the SRE and transcription is enhanced. If cholesterol levels rise, proteolytic cleavage of SREBP from the membrane ceases and any proteins in the nucleus are quickly degraded.

Question 37

statins

Answer 37

a class of drugs used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase. High cholesterol levels have been associated with cardiovascular disease (CVD). Statins have been found to prevent cardiovascular disease and mortality in those who are at high risk. The evidence is strong that statins are effective for treating CVD in the early stages of a disease (secondary prevention) and in those at elevated risk but without CVD (primary prevention). Side effects of statins include muscle pain, increased risk of diabetes and abnormalities in liver enzyme tests. Additionally, they have rare but severe adverse effects, particularly muscle damage

Question 38

sterol regulatory element binding protein (SREBP)

Answer 38

a protein that regulates both uptake and synthesis of cholesterol containing a transcription factor that regulates both LDLR and all 30 of the synthesis proteins. If cholesterol is low, the transcription factor is released, moves to the nucleus and activates all these genes. Since cholesterol is in membranes, the sensor to detect cholesterol is in the ER membrane, where cholesterol is lowest in the cell and changes would be easiest to detect. The transcription factor is a basic helix loop helix (bHLH) DNA- binding protein and is held inactive because it is part of a larger transmembrane protein (SREBP). The transcription factor only becomes active when it is cleaved from SREBP and then translocates to the nucleus. The proteases that cleave SREBP to release the bHLH (there are two of them) are located in the Golgi complex. SREBP must be held in the ER until cholesterol is low and then SREBP must move to the Golgi where it gets cleaved and the bHLH released.

Question 39

SCAP: SREBP cleavage activating protein

Answer 39

binds both SREBP and sterols like cholesterol. SCAP is a regulatory protein that is required for the proteolytic cleavage of the sterol regulatory element-binding protein (SREBP). SCAP is an integral membrane protein located in the endoplasmic reticulum (ER). One of the cytosolic regions of SCAP contains a hexapeptide amino acid sequence, MELADL, that functions to detect cellular cholesterol. When cholesterol is present, SCAP undergoes a conformational change that prevents it from activating SREBP and cholesterol synthesis does not occur. Scap has 8 transmembrane domains and both the N-terminal and C-terminal face the cytoplasm. Also, it binds SREBP by a series of consecutive WD repeats on its C-terminus.

Question 40

Insig

Answer 40

Insig binds SCAP only when cholesterol is high and when Insig binds, it blocks a signaling part of SCAP. This SCAP signal domain is recognized by a coat protein (COPII ) for vesicles that move from the ER to the Golgi. Thus, as cholesterol concentration drops, Insig no longer binds SCAP and the SCAP/SREBP complex gets packaged into vesicles to go to the Golgi

Question 41

RIP, regulated intramembrane proteolysis

Answer 41

One cleavage that releases bHLH cuts SREBP in the transmembrane domain. The prevailing dogma at the time of this discovery was that proteases cut proteins in an aqueous environment, not within a membrane. This cleavage within the membrane is now known as RIP, regulated intramembrane proteolysis. RIP has been shown to be critical also for Notch signaling in development and for cleavage of the amyloid precursor protein (APP) to produce the beta amyloid peptide in Alzheimer’s disease.

Question 42

cholesterol and heart attacks

Answer 42

It is well accepted that elevated cholesterol levels in plasma are linked to heart attacks. When LDL cholesterol levels are less than 100 mg/dl (total plasma cholesterol of 170 mg/dl), heart attacks are rare. What is perhaps less well appreciated is that these values of cholesterol for normal adults are typical of Western industrial societies with diets high in saturated animal fats and cholesterol. It is likely that the level of plasma cholesterol needed for normal physiology is much lower since animals and newborn humans have LDL cholesterol levels in the range of 25-50 mg/dl. These cholesterol levels are more in the expected range for the LDL receptor, which would have an optimal uptake for a plasma level about 25 mg/dl. These concepts have influenced new guidelines from the Adult Treatment Panel

Question 43

Membrane Proteins

Answer 43

Of the roughly 22,000 genes in the human genome, about one-tenth (2200) encode membrane transport proteins and ion channels. These are crucial (1) for getting nutrients into cells and organelles and (2) for signaling. A single one of these, the Na-K ATPase, uses about 30% of our ATP energy production to maintain the low concentration of Na+ and high concentration of K+ in cells. These proteins form aqueous pores through the membrane that are selective and regulated. In addition to transmembrane proteins, many proteins are membrane-associated. These can be attached to lipids or to the extracellular or intracellular domains of transmembrane proteins

Question 44

Various ways in which membrane proteins associate with the lipid bilayer.

Answer 44

Most transmembrane proteins are thought to extend across the bilayer as (1) a single α helix, (2) as multiple α helices, or (3) as a rolled-up β sheet (a β barrel). Some of these “single-pass” and “multipass” proteins have a covalently attached fatty acid chain inserted in the cytosolic lipid monolayer (1). Other membrane proteins are exposed at only one side of the membrane. (4) Some of these are anchored to the cytosolic surface by an amphipathic α helix that partitions into the cytosolic monolayer of the lipid bilayer through the hydrophobic face of the helix. (5) Others are attached to the bilayer solely by a covalently attached lipid chain—either a fatty acid chain or a prenyl group—in the cytosolic monolayer or, (6) via an oligosaccharide linker, to phosphatidylinositol in the noncytosolic monolayer. (7, 8) Finally, many proteins are attached to the membrane only by noncovalent interactions with other membrane proteins.

Question 45

glycosylphosphatidylinositol (GPI linkage)

Answer 45

A variety of proteins (e.g., several hydrolytic enzymes) are anchored to the membrane by glycosylphosphatidylinositol (GPI linkage). It is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification.This complex anchor to the membrane is found only on the exoplasmic/extracellular face. Some bacteria, e.g. Staphylococcus aureus and Listeria monocytogenes, secrete toxins that are soluble, bind to membranes and form pores to rupture membranes. One bacterium, Aeromonas hydrophilia, secretes a toxin that binds specifically to the GPI linkage, then inserts into the membrane, forms a pore and kills the cell.

Question 46

properties of transformed cells

Answer 46

altered morphology (rouded shape, refractile in phase contrast microscope), loss of contact inhibition (ability to grow over one another), ability to grow without attachment to solid substrate (anchorage independence), ability to proliferate indefinitely (immortalization), reduced requirements for mitogenic growth factors, high saturation density (ability to accumulate large numbers of cells in culture dish), inability to halt proliferation in response to deprivation of growth factors, increased transport of glucose (for anerobic respiration because of lack of oxygen), tumorigenicity

Question 47

acquired capabilities of cancer

Answer 47

self sufficency in growth signals, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis (growth of new blood vessels), evading apoptosis

Question 48

multi step tumorigenesis

Answer 48

tumor formation is really a multistep process, in which initially normal cell populations pass through a succession of intermediate stages on their way to becoming frankly malignant. Each of these intermediate stages contains cells that were more aberrant than those seen in the preceding steps. Quite possibly, each of these shifts in cell phenotype reflected a change in the underlying genetic makeup of the evolving premalignant cell population. Such a multistep genetic model of tumor progression stood in direct conflict with the single-hit model of transformation that was suggested by the discovery of the point-mutated ras oncogene.

Question 49

BCR-ABL

Answer 49

The exact chromosomal defect in Philadelphia chromosome is a translocation, in which parts of two chromosomes, 9 and 22, swap places. The result is that a fusion gene is created by juxtapositioning the Abl1 gene on chromosome 9 (region q34) to a part of the BCR (“breakpoint cluster region”) gene on chromosome 22 (region q11). This is a reciprocal translocation, creating an elongated chromosome 9 (der 9), and a truncated chromosome 22 (the Philadelphia chromosome). Because the Abl gene expresses a membrane-associated protein, a tyrosine kinase, the BCR-Abl transcript is also translated into a tyrosine kinase. The activity of tyrosine kinases is typically controlled by other molecules, but the mutant tyrosine kinase of the BCR-Abl transcript codes for a protein that is “always on” or continuously activated, which results in unregulated cell division (i.e. cancer). Although the BCR region also expresses serine/threonine kinases, the tyrosine kinase function is very relevant for drug therapy. Tyrosine kinase inhibitors (such as imatinib and sunitinib) are important drugs against a variety of cancers including CML, renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GISTs). Different fusions create different cancers (ALL, CML, CNL). In patients with BCR-ABL translocation (The Philadelphia chromosome), the ABL tyrosine kinase can be inhibited by Gleevac (ST1571), an ATP analogue. Clinical trials have shown that Gleevac (ST1571) is effective in 95% of these patients!

Question 50

Loss of heterozygosity (LOH)

Answer 50

a gross chromosomal event that results in loss of the entire gene and the surrounding chromosomal region. The loss of heterozygosity is a common occurrence in cancer, where it indicates the absence of a functional tumor suppressor gene in the lost region. However, many people remain healthy with such a loss, because there still is one functional gene left on the other chromosome of the chromosome pair. However, the remaining copy of the tumor suppressor gene can be inactivated by a point mutation, leaving no tumor suppressor gene to protect the body. Loss of heterozygosity does not imply a reversal to the homozygous state. The classical example of such a loss of protecting genes is hereditary retinoblastoma, in which one parent’s contribution of the tumor suppressor Rb1 is flawed. Although most cells will have a functional second copy, chance loss of heterozygosity events in individual cells almost invariably lead to the development of this retinal cancer in the young child. Loss of heterozygosity can be identified in cancers by noting the presence of heterozygosity at a genetic locus in an organism’s germline DNA, and the absence of heterozygosity at that locus in the cancer cells. This is often done using polymorphic markers, such as microsatellites or single-nucleotide polymorphisms, for which the two parents contributed different alleles. Genome-wide LOH status of fresh or paraffin embedded tissue samples can be assessed by virtual karyotyping using SNP arrays.

Question 51

Knudson two-hit hypothesis of tumorigenesis

Answer 51

First Hit: The first hit is classically thought of as a point mutation that inactivates one copy of a tumor suppressor gene (TSG), such as Rb1. In hereditary cancer syndromes, individuals are born with the first hit. The individual does not develop cancer at this point because the remaining TSG allele on the other locus is still functioning normally. Second Hit: While the second hit is commonly assumed to be a deletion that results in loss of the remaining functioning TSG allele, the original published mechanism of RB1 LOH was mitotic recombination/gene conversion/copy-neutral LOH, not deletion. There is a critical difference between deletion and CN-LOH (copy neutral-LOH), as the latter mechanism cannot be detected by comparative genomic hybridization (CGH)-based gene copy number counting, and requires allelic genotyping. Either way, LOH leaves only non-functioning alleles of the TSG, and the individual goes on to develop cancer.

Question 52

familial retinoblastoma

Answer 52

a rapidly developing cancer that develops from the immature cells of a retina, the light-detecting tissue of the eye and is the most common malignant tumor of the eye in children. In most children with retinoblastoma, the disease affects only one eye. However, one out of three children with retinoblastoma develops cancer in both eyes. The most common first sign of retinoblastoma is a visible whiteness in the pupil called “cat’s eye reflex” or leukocoria. This unusual whiteness is particularly noticeable in photographs taken with a flash. Other signs and symptoms of retinoblastoma include crossed eyes or eyes that do not point in the same direction (strabismus); persistent eye pain, redness, or irritation; and blindness or poor vision in the affected eye(s). In germinal retinoblastoma, mutations in the RB1 gene appear to be inherited in an autosomal dominant pattern. When retinoblastoma is associated with a gene mutation that occurs in all of the body’s cells, it is known as germinal retinoblastoma. People with this form of retinoblastoma also have an increased risk of developing several other cancers outside the eye. Specifically, they are more likely to develop a cancer of the pineal gland in the brain (pinealoma), a type of bone cancer known as osteosarcoma, cancers of soft tissues such as muscle, and an aggressive form of skin cancer called melanoma.

Question 53

inactivation of Rb

Answer 53

Rb is phosphorylated to pRb by certain Cyclin Dependent Kinases (CDKs). pRb is described as being hyperphosphorylated and when in this state, it is unable to complex E2F and therefore, unable to restrict progression from the G1 phase to the S phase of the cell cycle. During the M-to-G1 transition, pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypophosphorylated state Rb . When it is time for a cell to enter S phase, complexes of cyclin-dependent kinases (CDK) and cyclins phosphorylate Rb to pRb, inhibiting its activity. The initial phosphorylation is performed by Cyclin D/CDK4/CDK6 and followed by additional phosphorylation by Cyclin E/CDK2. pRb remains phosphorylated throughout S, G2 and M phases. Phosphorylation of Rb allows E2F-DP to dissociate from pRb and become active. When E2F is free it activates factors like cyclins (e.g. Cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or PCNA, which speeds DNA replication and repair by helping to attach polymerase to DNA

Question 54

tumor viruses

Answer 54

Tumor viruses come in a variety of forms: viruses with a DNA genome, such as adenovirus, and viruses with an RNA genome, like the Hepatitis C virus (HCV) can cause cancers, as can retroviruses having both DNA and RNA genomes (Human T-lymphotropic virus and hepatitis B virus, which normally replicates as a mixed double and single-stranded DNA virus but also has a retroviral replication component). In many cases, tumor viruses do not cause cancer in their native hosts but only in dead-end species. For example, adenoviruses do not cause cancer in humans but are instead responsible for colds, conjunctivitis and other acute illnesses. They only become tumorigenic when infected into certain rodent species, such as Syrian hamsters. Some viruses are tumorigenic when they infect a cell and persist as circular episomes or plasmids, replicating separately from host cell DNA (Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus). Other viruses are only carcinogenic when they integrate into the host cell genome as part of a biological accident, such as polyomaviruses and papillomaviruses. A direct oncogenic viral mechanism involves either insertion of additional viral oncogenic genes into the host cell or to enhance already existing oncogenic genes (proto-oncogenes) in the genome. Indirect viral oncogenicity involves chronic nonspecific inflammation occurring over decades of infection, as is the case for HCV-induced liver cancer. These two mechanisms differ in their biology and epidemiology: direct tumor viruses must have at least one virus copy in every tumor cell expressing at least one protein or RNA that is causing the cell to become cancerous. Because foreign virus antigens are expressed in these tumors, persons who are immunosuppressed such as AIDS or transplant patients are at higher risk for these types of cancers. Chronic indirect tumor viruses, on the other hand, can be lost (at least theoretically) from a mature tumor that has accumulated sufficient mutations and growth conditions (hyperplasia) from the chronic inflammation of viral infection. In this latter case, it is controversial but at least theoretically possible that an indirect tumor virus could undergo “hit-and-run” and so the virus would be lost from the clinically diagnosed tumor. In practical terms, this is an uncommon occurrence if it does occur.

Question 55

activation of Rb

Answer 55

When it is time for a cell to enter S phase, complexes of cyclin-dependent kinases (CDK) and cyclins phosphorylate Rb to pRb, inhibiting its activity.[2][3][18][19] The initial phosphorylation is performed by Cyclin D/CDK4/CDK6 and followed by additional phosphorylation by Cyclin E/CDK2. pRb remains phosphorylated throughout S, G2 and M phases.

Question 56

E7 protein

Answer 56

produced by HPV and phosphorlates Rb. E7 protein has both transforming and trans-activating activities. Disrupts the function of host retinoblastoma protein RB1/pRb, which is a key regulator of the cell cycle. Induces the disassembly of the E2F1 transcription factors from RB1, with subsequent transcriptional activation of E2F1-regulated S-phase genes. Inactivation of the ability of RB1 to arrest the cell cycle is critical for cellular transformation, uncontrolled cellular growth and proliferation induced by viral infection. Stimulation of progression from G1 to S phase allows the virus to efficiently use the cellular DNA replicating machinery to achieve viral genome replication.

Question 57

E6 protein

Answer 57

The E6 protein is a major transforming protein of many types of papillomaviruses. Mechanistically, the best characterized E6 proteins are those of the high-risk genital HPVs (e.g. HPV-16 and 18 E6), which function, at least in part, by inactivating the p53 tumor suppressor protein. Biochemical studies have shown that this occurs by targeted degradation of p53, dependent on the E6-AP ubiquitin-protein ligase. Since the expression of E6 is strictly required for maintenance of a malignant phenotype in HPV-induced cancers, it is an appealing target of therapeutic HPV vaccines designed to eradicate established cervical cancer tumors.

Question 58

Large T protein

Answer 58

produced by SV40 (monkey virus) and inactivates Rb and p53. a hexamer protein that is a proto-oncogene derived from the polyomavirus SV40 which is capable of transforming a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the retinoblastoma (pRB) and p53 tumor suppressor proteins. In addition, TAg binds to several other cellular factors, including the transcriptional co-activators p300 and CBP, which may contribute to its transformation function

Question 59

Kaposi’s sarcoma-associated herpesvirus (KSHV)

Answer 59

This virus causes Kaposi’s sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma and some types of multicentric Castleman’s disease. It is one of seven currently known human cancer viruses, or oncoviruses. KSHV is a herpesvirus

Question 60

herpesviruses

Answer 60

a large family of DNA viruses that cause diseases in animals, including humans. The family name is derived from the Greek word herpein (“to creep”), referring to the latent, recurring infections typical of this group of viruses. Herpesviridae can cause latent or lytic infections.

Question 61

p107

Answer 61

The protein encoded by this gene is similar in sequence and possibly function to the product of the retinoblastoma 1 (RB1) gene. The RB1 gene product is a tumor suppressor protein that appears to be involved in cell cycle regulation, as it is phosphorylated in the S to M phase transition and is dephosphorylated in the G1 phase of the cell cycle. Both the RB1 protein and the product of this gene can form a complex with adenovirus E1A protein and SV40 Large T-antigen, with the SV40 large T-antigen binding only to the unphosphorylated form of each protein. In addition, both proteins can inhibit the transcription of cell cycle genes containing E2F binding sites in their promoters. Due to the sequence and biochemical similarities with the RB1 protein, it is thought that the protein encoded by this gene may also be a tumor suppressor.

Question 62

p130

Answer 62

p130 is a tumor suppressor of the pocket protein family whose expression is posttranscriptionally regulated and largely G0 restricted. The mechanism of down-regulation of p130 expression in proliferating cells was investigated. Our results indicate that the decline of p130 expression as G0 cells reenter the cell cycle is due to a decrease in protein stability.

Question 63

Adenomatous polyposis coli (APC)

Answer 63

The APC protein is a negative regulator that controls Beta-catenin concentrations and interacts with E-cadherin, which are involved in cell adhesion. The most common mutation in colon cancer is inactivation of APC. When APC does not have an inactivating mutation, frequently there are activating mutations in beta catenin. Mutations in APC can be inherited, or arise sporadically in the somatic cells, often as the result of mutations in other genes that result in the inability to repair mutations in the DNA. In order for cancer to develop, both alleles (copies of the APC gene) must be mutated.

Question 64

Wnt signaling pathways

Answer 64

the Wnt pathway that causes an accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family. Without Wnt signaling, the β-catenin would not accumulate in the cytoplasm since a destruction complex would normally degrade it. This destruction complex includes the following proteins: Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α). It degrades β-catenin by targeting it for ubiquitination, which subsequently sends it to the proteasome to be digested. However, as soon as Wnt binds Fz and LRP-5/6, the destruction complex function becomes disrupted. This is due to Wnt causing the translocation of the negative Wnt regulator, Axin, and the destruction complex to the plasma membrane. Phosphorylation by other proteins in the destruction complex subsequently binds Axin to the cytoplasmic tail of LRP-5/6. Axin becomes de-phosphorylated and its stability and levels are decreased. Dsh then becomes activated via phosphorylation and its DIX and PDZ domains inhibit the GSK3 activity of the destruction complex. This allows β-catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF (T-cell factor/lymphoid enhancing factor) transcription factors, leading to transcription of c-myc. Increased β-catenin expression is strongly correlated with poor prognosis in breast cancer patients. This accumulation may be due to several factors such as mutations in β-catenin, deficiencies in the β-catenin destruction complex, most frequently by mutations in structurally disordered regions of APC, overexpression of Wnt ligands, loss of inhibitors, and/or decreased activity of regulatory pathways (such as the Wnt/calcium pathway).

Question 65

BRCA1

Answer 65

BRCA1 is a human tumor suppressor gene. BRCA1 combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome surveillance complex (BASC). The BRCA1 protein associates with RNA polymerase II, and through the C-terminal domain, also interacts with histone deacetylase complexes. Thus, this protein plays a role in transcription, DNA repair of double-strand breaks[9] ubiquitination, transcriptional regulation as well as other functions.

Question 66

BRCA2

Answer 66

BRCA2 binds the single strand DNA and directly interacts with the recombinase RAD51 to stimulate strand invasion a vital step of homologous recombination. The localization of RAD51 to the DNA double-strand break requires the formation of BRCA1-PALB2-BRCA2 complex. PALB2 (Partner and localizer of BRCA2) can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. These breaks can be caused by natural and medical radiation or other environmental exposures, but also occur when chromosomes exchange genetic material during a special type of cell division that creates sperm and eggs (meiosis). Double strand breaks are also generated during repair of DNA cross links. By repairing DNA, these proteins play a role in maintaining the stability of the human genome and prevent dangerous gene rearrangements that can lead to hematologic and other cancers. Recently, BRCA2 has been shown to be allelic with the Fanconi’s anemia D1 gene, FANCD1. This means that individuals with homozygous mutations in BRCA2 get Fanconi’s anemia, while heterozygotes get breast cancer from rare recombinant cells in the mammary gland that lose the wild-type allele.

Question 67

p53

Answer 67

The p53 gene is a very, important cancer predisposing gene because mutant p53 is found in about 50% of all cancers! p53 is a tumor suppressor gene. It also is important for the response of cells to environmental mutagenesis. Cells missing p53 accumulate mutations at a much higher rate and thereby, have a greater chance of becoming malignant. In this way, p53 acts as a “guardian of the genome” preventing potentially deleterious mutations. The p53 gene was initially found to be an oncogene, in that certain p53 mutant genes were dominant to the wild-type gene in producing cellular transformation. Later studies indicated that this gene behaved as a classic tumor suppressor in Li-Fraumeni syndrome. The explanation was found by showing that the oncogenic p53 mutations produce a mutant p53 protein that can bind the wild-type p53 protein and inactivate it. These “dominant-negative” p53 mutations can be viewed as “spoilers” or “monkey wrenches”. p53 protein acts as a transcription factor important for the expression of genes, which prevent cells from replicating damaged or foreign DNA. p53 is also required for apoptosis, in which cells commit suicide if their DNA is damaged beyond repair. In p53 defective cells, damaged DNA is replicated, thereby producing additional mutations including chromosomal rearrangements, which can lead to cancer. In this manner, p53 acts as a “guardian of the genome”. Certain p53 point mutations are found more frequently than others in human cancers. These mutations are called “hotspots”. For example, mutational “hotspots” produce alterations in amino acids 248 or 273 of p53 in all human cancers. Some “hotspots” are unique to specific cancers. For example, an alteration in amino-acid 157 of p53 is found mainly in lung cancer and is the result of the mutagenic chemicals found in cigarette smoke. p53 interferes with the life cycle of many human viruses including Adenovirus and HPV (human papilloma virus). The viruses have oncogenes that act by inactivating p53, for example, Adenovirus E1B and HPV E6 proteins. Remember that these viruses also inactivate RB protein. In fact, destruction of both RB and p53 either by cellular mutations or viruses is a major route to cancer. The most common mutation is in the DNA binding region. Most of these mutations destroy the ability of the protein to bind to its target DNA sequences, and thus prevents transcriptional activation of these genes, which is usually a recessive loss of function mutation (not in the case of p53).

Question 68

Oncogenes

Answer 68

Oncogenes were discovered in certain oncogenic retroviruses. Although some have nearly the same DNA sequences as the cognate v-onc gene most are quite different. Thus, if v-onc genes originated from c-onc genes, substantial rearrangements occurred during or after the capture (at least with many such oncogenes). The process of activation of proto-oncogenes to oncogenes can include retroviral transduction or retroviral integration (see below), point mutations, insertion mutations, gene amplification, chromosomal translocation and/or protein-protein interactions.A few c-onc genes ligated to retroviral promoters can transform normal cells when introduced via DNA mediated transformation (examples: c-ras). Other c-onc genes do not directly transform normal cells. Translocations of c-onc genes have been observed in certain human cancers, and also indicate a poor prognosis. The translocation results in inappropriate and high level expression of the oncogene (See Burkitt lymphoma and the Philadelphia chromosome (BCR-ABL translocation).

Question 69

Retroviruses

Answer 69

Retroviruses are single strand RNA containing membrane enclosed viruses that bud from the cell membrane of infected cells and which usually do not kill the infected cell. The RNA genome consists of two identical strands held together by a tRNA molecule. The gag gene codes for internal virion proteins, the env gene for virus membrane glycoproteins, and the pol gene for a virus polymerase. When these genes are the only ones present the virus does not cause tumors, but replicates through an intermediate proviral DNA and integrates into the host cell genome. It can be transmitted as an integrated provirus through somatic or sex cells as a cellular gene.

Question 70

src gene

Answer 70

a non-receptor protein tyrosine kinase protein that in humans is encoded by the SRC gene. This protein phosphorylates specific tyrosine residues in other proteins. An elevated level of activity of c-Src tyrosine kinase is suggested to be linked to cancer progression by promoting other signals. c-Src includes an SH2 domain, an SH3 domain, and a tyrosine kinase domain. c-Src can be activated by many transmembrane proteins that include: adhesion receptors, receptor tyrosine kinases, G-protein coupled receptors and cytokine receptors. Most studies have looked at the receptor tyrosine kinases and examples of these are platelet derived growth factor receptor (PDGFR) pathway and epidermal growth factor receptor (EGFR). When src is activated, it promotes survival, angiogenesis, proliferation and invasion pathways. The activation of the c-Src pathway has been observed in about 50% of tumors from colon, liver, lung, breast and the pancreas. Since the activation of c-Src leads to the promotion of survival, angiogenesis, proliferation and invasion pathways, the aberrant growth of tumors in cancers is observed. A common mechanism is that there are genetic mutations that result in the increased activity or the overexpression of the c-Src leading to the constant activation of the c-Src. A number of tyrosine kinase inhibitors that target c-Src tyrosine kinase (as well as related tyrosine kinases) have been developed for therapeutic use. One notable example is dasatinib which has been approved for the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (PH+) acute lymphocytic leukemia (ALL).

Question 71

v-oncogenes

Answer 71

a virus that can cause cancer. Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells.

Question 72

Retroviruses life cycle

Answer 72

The retroviral life cycle begins in the nucleus of an infected cell. At this stage of the life cycle the retroviral genome is a DNA element integrated into and covalently attached to the DNA of the host cell.The genome of the virus is of approximately 8-12 kilobases of DNA (depending upon the retroviral species). Full-length genomic mRNA is made initiating at the beginning ofthe R (repeat) at the 5’ LTR (Long Terminal Repeat).The free particle can infect new cells by binding to a cell surface receptor. The specificity of the virus-cell interaction is determined largely by the envelope protein(s) of the retrovirus. Infection leads to injection of the virus nucleoprotein core (consisting mostly of gag-derived proteins, full-length genomic RNA, and the reverse transcriptase protein). Once inside the cell, the nucleoprotein complex accesses intracellular DNA nucleotide triphosphate pools, whereupon the reverse transcriptase protein initiates creation of a double-stranded DNA copy of the genome of the virus in preparation for integration into the host cell chromosome. Upon completion of reverse transcription, the viral enzyme Integrase searches the DNA for an appropriate “home”, whereupon the integrase clips the host DNA and sews the double-stranded DNA into the host DNA (see below). The virus is now prepared to initiate a new round of replication.

Question 73

ALV virion to RSV virion

Answer 73

Avian sarcoma leukosis virus (ASLV) is an endogenous retrovirus that infects and can lead to cancer in chickens; experimentally it can infect other species of birds and mammals. ASLV is genetically closely related to the Rous sarcoma virus (RSV), but unlike RSV, ASLV does not contain the src gene, which codes for a tyrosine kinase, and does not transform the fibroblasts that it infects.[2] Both RSV and ASLV contain the gag gene, which is common to most retroviruses and encodes for the capsid proteins, and the pol gene which encodes for the reverse transcriptase enzyme. ASLV and some RSVs also contain the env gene, which encodes a precursor polyprotein that assembles in the endoplasmic reticulum. The polyproteins are then transported to the Golgi apparatus, glycosylated and cleaved to produce two glycoproteins: one surface and one transmembrane

Question 74

phosphoglyceride

Answer 74

glycerol-based phospholipids.

Question 75

Why should I care about lipids?

Why should you care about lipids?

Answer 75

Proteins get most of the attention because they pump ions, transport nutrients, and act as receptors for hormones. We get the idea that these greasy lipid molecules simply form a passive support for proteins. Wrong. Yes, they form the bilayer, but they are crucial for many functions. (1) Transmembrane proteins have to interact with lipids to form the right shape in the membrane. (2) Lipids interact with proteins to hold them in clusters. (3) Lipids act as signaling molecules (esp. phosphoinositides). (4) Movement of PS from the inner to outer leaflet will tell macrophages to “kill that cell.” Lipids are not just inert molecules.

Question 76

site of lipid synthesis

Answer 76

Most lipids are synthesized in ER and some are finished in the Golgi complex. Ceramide plus a sugar is a glycosphingolipid. When sialic acids are added, these are gangliosides; there are at least 60 different ones depending on how the sugars and sialic acids are attached. Inability to degrade these leads to storage diseases like Tay Sachs Disease.

Question 77

sphingomyelin

Answer 77

a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphocholine and ceramide, or a phosphoethanolamine head group; therefore, sphingomyelins can also be classified as sphingophospholipids.

Question 78

glucosylceramide

Answer 78

any of the cerebrosides in which the monosaccharide head group is glucose. They occur mostly in nonneuronal tissue and accumulate abnormally in Gaucher disease, where glucocerebrosidase is absent or nonfunctional.

Question 79

Low-Density Lipoprotein (LDL) Receptor

Answer 79

LDL receptor complexes are present in clathrin-coated pits (or buds) on the cell surface, which when bound to LDL-cholesterol via adaptin, are pinched off to form clathrin-coated vesicles inside the cell. This allows LDL-cholesterol to be bound and internalized in a process known as endocytosis and prevents the LDL just diffusing around the membrane surface. Once the coated vesicle is internalized it will shed its clathrin coat and will fuse with an acidic late endosome. The change in pH causes a conformational change in the receptor that releases the bound LDL particle. The receptors are then either destroyed or they can be recycled via the endocytic cycle back to the surface of the cell where the neutral pH will cause the receptor to revert to its native conformation ready to receive another LDL particle. Synthesis of receptors in the cell is regulated by the level of free intracellular cholesterol; if it is in excess for the needs of the cell then the transcription of the receptor gene will be inhibited. LDL receptors are translated by ribosomes on the endoplasmic reticulum and are modified by the Golgi apparatus before travelling in vesicles to the cell surface.

Question 80

Sec24

Answer 80

The protein encoded by this gene is a member of the SEC24 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec24p component of COPII. COPII is the coat protein complex responsible for vesicle budding from the ER. The role of this gene product is implicated in the shaping of the vesicle, and also in cargo selection and concentration. Two transcript variants encoding different isoforms have been found for this gene.

Question 81

Exoplasmic face

Answer 81

PC, sphingomyelin, and glycolipids are more abundant on the external surface.

Question 82

Flipase

Answer 82

a family of transmembrane lipid transporter enzymes located in the membrane responsible for aiding the movement of phospholipid molecules between the two leaflets that compose a cell’s membrane (transverse diffusion). Although phospholipids diffuse rapidly in the plane of the membrane, their polar head groups cannot pass easily through the hydrophobic center of the bilayer, limiting their diffusion in this dimension. Phospholipid molecules that are synthesized in the cell are incorporated into the cytoplasmic face of the membrane, where flippases can transfer them to the exoplasmic face. Energy-dependent flippases require energy input in the form of ATP to carry out their function, often known as a flip-flop. However, there are energy-independent flippases that do not require the hydrolysis of ATP and are unidirectional in their action. These energy-independent flippases are responsible for transferring newly synthesised lipids from the outer to the inner leaflet of membranes. Many cells maintain asymmetric distributions of phospholipids between their cytoplasmic and exoplasmic membrane leaflets.[2] The loss of asymmetry, in particular the appearance of the anionic phospholipid phosphatidylserine on the exoplasmic face, can serve as an early indicator of apoptosis.[3] This effect has been observed in neurons as a response to amyloid beta peptides, thought to be a primary cause of the neurodegenerative effects of Alzheimer’s disease.[

Question 83

Bilayer curvature

Answer 83

Membrane curvature is dependent on size of the head group vs. the non-polar tail; it is also dependent on protein composition.

Question 84

Listeria

Answer 84

Listeriosis, a serious infection usually caused by eating food contaminated with the bacterium Listeria monocytogenes, is an important public health problem in the United States. The disease primarily affects older adults, pregnant women, newborns, and adults with weakened immune systems.

Question 85

c-src

Answer 85

is a non-receptor protein tyrosine kinase protein that in humans is encoded by the SRC gene. This protein phosphorylates specific tyrosine residues in other proteins. An elevated level of activity of c-Src tyrosine kinase is suggested to be linked to cancer progression by promoting other signals. c-src has a different carboxy-terminal amino acid sequence that v-src and has numerous introns that do not exist in v-src.

Question 86

c-myc

Answer 86

a regulator gene that codes for a transcription factor. The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. A mutated version of Myc is found in many cancers, which causes Myc to be constitutively (persistently) expressed. This leads to the unregulated expression of many genes, some of which are involved in cell proliferation, and results in the formation of cancer.[1] A common human translocation involving Myc is critical to the development of most cases of Burkitt’s Lymphoma. c-myc also has many introns not present in v-myc, although the coding sequences are nearly identical (7 amino acid changes). N-myc, a member of the c-myc family of oncogenes, is found amplified in neuroblastoma.

Question 87

c-ras

Answer 87

Ras proteins function as binary molecular switches that control intracellular signaling networks. Ras-regulated signal pathways control such processes as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. Ras and ras-related proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis. Ras activates several pathways, of which the mitogen-activated protein (MAP) kinase cascade has been well-studied. This cascade transmits signals downstream and results in the transcription of genes involved in cell growth and division. There is a separate AKT pathway that inhibits apoptosis. Analysis of DNA from human bladder cancer cells has shown that c-ras genes have been point mutated, supporting the qualitative model (overactive or unregulated protein). Mutations are found in either codon 12 or codon 61 of the ras gene product. These mutations produce a ras protein that is unregulated and is always “on”. Detection of these ras mutations indicates a poor prognosis.

Question 88

HER2/neu oncogene (also called erbB2),

Answer 88

HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or over-expression of the ERBB2 gene occurs in approximately 15-30% of breast cancers. It is strongly associated with increased disease recurrence and a poor prognosis. Over-expression is also known to occur in ovarian, stomach, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. The HER2/neu oncogene (also called erbB2), which encodes a integral membrane protein kinase (see v-erb-B) is amplified in about 20% of breast cancers. Higher levels of amplification correlate with a poor prognosis. This supports the quantitative model above. Drugs such as monoclonal antibodies specific for the protein product of the HER2/neu/erbB2 oncogene can reverse the transformed phenotype of the cancer cell. These antibodies are called Herceptin (See http://www.herceptin.com/ for more information). Furthermore, drugs that inhibit HER2/neu/erbB2 are being used to extend the life of breast cancer patients. More drugs of this type are currently in clinical trials for both breast and lung cancer.

Question 89

Cancer “Targeted” Therapy

Answer 89

In theory, tumor cells may have some of their properties reversed by either blocking the action of oncogenes and/or adding back any missing tumor suppressors. This can be accomplished either at the gene level (gene therapy) or by using drugs or antibodies. Antigens that are involved in growth and differentiation signaling are often growth factors and growth factor receptors. Growth factors are targets for antibodies in cancer patients. The goal is to identify the defect in the tumor from the patient and then targeting the therapy to fit it. This is called “Molecularly targeted therapy” or “rationally designed drug therapy. Why do drugs that inhibit “normal” cellular proteins (c-myc, c-abl, etc.) kill only the cancer cells? One idea is that cancer cells but not normal cells have become dependent or “addicted” to the overexpressed oncogene. This referred to as “oncogene addiction”. In summary, oncogenes and tumor suppressor genes are being used as “molecular markers” in both cancer diagnosis and prognosis. The elucidation of the function of these genes also will be useful for therapies, either at the gene and/or protein levels and forms the basis of “Personalized Medicine”.

Question 90

Herceptin

Answer 90

a monoclonal antibody that interferes with the HER2/neu receptor. Its main use is to treat certain breast cancers. The HER receptors are proteins that are embedded in the cell membrane and communicate molecular signals from outside the cell (molecules called EGFs) to inside the cell, and turn genes on and off. The HER proteins stimulate cell proliferation. In some cancers, notably certain types of breast cancer, HER2 is over-expressed, and causes cancer cells to reproduce uncontrollably. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is reduced proliferation. It has been suggested that trastuzumab induces some of its effect by downregulation of HER2/neu leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade.[8] In addition, trastuzumab suppresses angiogenesis both by induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated growth observed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. Trastuzumab has been shown to inhibit HER2/neu ectodomain cleavage in breast cancer cells.

Question 91

p53 cancer targeted treatment

Answer 91

Injection of the RB gene into a RB-negative small cell lung cancer line will inhibit tumorigenesis. E1B mutant adenoviruses, which cannot inactivate p53 and thereby cannot kill wild-type cells, will kill p53- cancer cells preferentially . A drug has been used to correct the mutant conformation of dominant-negative p53 mutations . This drug blocks the production of tumors by these p53 mutant cells in a mouse model system.

Question 92

Personalized Medicine and Cancer

Answer 92

Molecular knowledge of the genome in cancer cells (Bioinformatics) is being used for targeted therapy and for “personalized medicine” in cancer. In an example of this type of procedure, a “heat map” is created in which changes in gene copy number are correlated with tumor grade. The “heat map” is created by hybridization of the tumor DNA to “gene chip” containing all human genomic DNA sequences as molecular probes. Red color indicates increases and green indicates decreases. It is called a “heat map” because it resembles graphical representation of hot and cold areas of the earth. With the advent of whole genome sequencing, cancer therapy is being designed on a personal basis depending on the type of mutations (oncogene, tumor suppressor, etc.) in the tumor and the known therapeutic responses to these lesions.

Question 93

heat maps

Answer 93

shows you differential expression by displaying gene expression values in a heat map format. Each colored cell in the heat map represents the gene expression value for a probe in a sample. The largest gene expression values are displayed in red (hot), the smallest values in blue (cool), and intermediate values in shades of red (pink) or blue.“Heat maps’ of this type are being used to correlate many types of molecular data (gene copy number, gene expression, heat maps, mutations, etc.) with relevant clinical information (tumor grade, survival, age, tumor stage, etc.). In breast cancer, this type of analysis is already being used in the clinic. The Breast cancer Xpress Chip measures the expression of 123 genes known to be altered in breast cancers. This includes tumor suppressors such as BRCA1 and p53 and oncogenes such as estrogen receptor (ER) and erbB2. The information is then used for diagnosis, prognosis and therapy. For example, patients with high erbB2 are treated with Herceptin and those with high ER levels are treated with tamoxifen (estrogen antagonist).

Question 94

HIV Entry into Cells

Answer 94

To infect a cell, a virus must first be able to enter it. HIV is an enveloped virus and accomplishes cell entry by fusing the viral membrane with the cellular plasma membrane. This process is carried out by the viral envelope proteins gp120 and gp41. These two proteins are synthesized as a single 160 kD protein which is then cleaved. The products of this cleavage remain associated until the process of viral entry into the cell begins. gp120 binds to CD4 on CD4+ T lymphocytes and cells of the monocyte/macrophage lineage. This binding event and further interaction between gp120 and cellular co-receptors lead to gp120 dissociation from gp41. The dissociation of gp120 occurs as part of a conformational change in gp41 that leaves it in a “fusion-active” form. This form of gp41 can then mediate fusion between the cellular and viral membranes. Since a number of both viral and cellular proteins that carry out membrane fusion have similar structures, it is thought that the mechanisms of fusion may be similar in these different systems. The common structure is that of a helical coiled coil, in which three alpha helices wrap around each other. In the fusion-active state of many of these molecules, the helices undergo a reversal partway through the coiled coil and bend back on themselves, packing along the outside of the initial coil. Fusion is generally thought to occur via the formation of a pore between two closely apposed membranes. In its pre-fusion hairpin conformation, the fusion peptide is thought to be located in the target membrane, while the transmembrane region is in the viral membrane.