cell biology 3

Question 1

DNA Checkpoint Regulation

Answer 1

usually occurs by activation of specific enzymes that modify and inhibit proteins needed for cell cycle progression. In the DNA damage checkpoint, CDK cyclin complexes are inhibited by p53 and Chk2. Patients with Li Fraumeni syndrome have a high risk for cancer and have mutated p53 or Chk2 gene. The idea is that these patients accumulate many mutations leading to cancer.

Question 2

Li Fraumeni syndrome

Answer 2

greatly increases susceptibility to cancer. This syndrome is also known as the Sarcoma, breast, leukaemia and adrenal gland (SBLA) syndrome. The syndrome is linked to germline mutations of the TP53 tumor suppressor gene, which normally helps control cell growth. The TP53 (tumor suppressor gene p53) normally assists in the control of cell division and growth through action on the normal cell cycle. TP53 assists in repair or destruction of “bad” DNA before it can enter the normal cell cycle, thus preventing abnormal and/or cancerous growth of cells. Mutations of TP53 prevent this normal function and allow damaged cells to divide and grow in an uncontrolled, unchecked manner forming tumors (cancers).

Question 3

Chk2

Answer 3

is a protein kinase that is activated in response to DNA damage and is involved in cell cycle arrest. It is rapidly phosphorylated (by both ATR and ATM) in response to replication blocks and DNA damage. When activated, the protein is known to inhibit CDC25C phosphatase, preventing entry into mitosis, and has been shown to stabilize the tumor suppressor protein p53, leading to cell cycle arrest in G1

Question 4

p53

Answer 4

P53 gene is located on the short arm of chromosome 17 (17p13.1). is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor, preventing cancer. It is a sequence specific DNA binding protein, which activates the transcription of genes in cell cycle assert and cell death. It is downregulated by binding to the MDM2 protein (expression is also controlled by p53, negative feedback loop) which not only masks its activation domain, but also targets it for destruction by the ubiquitin-proteasome pathway. It is an example of regulating amount of transcription factor in the cell. It is mutated in about 50% of cancers. Different properties of each mutation may in part be explained by clinical heterogeneity. Some mutations are more common in families others are more common in sporadic tumors

Question 5

mutated p53

Answer 5

common in cancer of all kinds, in tumors with mutated p53, there is actualy a high level of MDM2 because p53 activates transcription of MDM2, but it also makes it dominent negative, inhibiting the wildtype protein

Question 6

dominant negative mutation

Answer 6

A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell, usually by dimerizing (combining) with it. In cases of polymeric molecules, such as collagen, dominant negative mutations are often more deleterious than mutations causing the production of no gene product (null mutations or null alleles).

Question 7

MDM2 protein

Answer 7

an important negative regulator of the p53 tumor suppressor.

Question 8

Signs and symptoms of DKA

Answer 8

The symptoms of an episode of diabetic ketoacidosis usually evolve over the period of about 24 hours. Predominant symptoms are nausea and vomiting, pronounced thirst, excessive urine production and abdominal pain that may be severe. Those who measure their glucose levels themselves may notice hyperglycemia (high blood sugar levels). In severe DKA, breathing becomes labored and of a deep, gasping character (a state referred to as “Kussmaul respiration”). The abdomen may be tender to the point that an acute abdomen may be suspected, such as acute pancreatitis, appendicitis or gastrointestinal perforation. Coffee ground vomiting (vomiting of altered blood) occurs in a minority of patients; this tends to originate from erosion of the esophagus. In severe DKA, there may be confusion, lethargy, stupor or even coma (a marked decrease in the level of consciousness). On physical examination there is usually clinical evidence of dehydration, such as a dry mouth and decreased skin turgor. If the dehydration is profound enough to cause a decrease in the circulating blood volume, tachycardia (a fast heart rate) and low blood pressure may be observed. Often, a “ketotic” odor is present, which is often described as “fruity”, often compared to the smell of pear drops whose scent is a ketone. If Kussmaul respiration is present, this is reflected in an increased respiratory rate. Small children with DKA are relatively prone to cerebral edema (swelling of the brain tissue), which may cause headache, coma, loss of the pupillary light reflex, and progress to death. It occurs in 0.3–1.0% of children with DKA, and has been described in young adults, but is overall very rare in adults.

Question 9

mechanism of DKA

Answer 9

Diabetic ketoacidosis arises because of a lack of insulin in the body. The lack of insulin and corresponding elevation of glucagon leads to increased release of glucose by the liver (a process that is normally suppressed by insulin) from glycogen via glycogenolysis and also through gluconeogenesis. High glucose levels spill over into the urine, taking water and solutes (such as sodium and potassium) along with it in a process known as osmotic diuresis. This leads to polyuria, dehydration, and compensatory thirst and polydipsia. The absence of insulin also leads to the release of free fatty acids from adipose tissue (lipolysis), which are converted, again in the liver, into ketone bodies (acetoacetate and β-hydroxybutyrate). β-Hydroxybutyrate can serve as an energy source in the absence of insulin-mediated glucose delivery, and is a protective mechanism in case of starvation. The ketone bodies, however, have a low pKa and therefore turn the blood acidic (metabolic acidosis). The body initially buffers the change with the bicarbonate buffering system, but this system is quickly overwhelmed and other mechanisms must work to compensate for the acidosis. One such mechanism is hyperventilation to lower the blood carbon dioxide levels (a form of compensatory respiratory alkalosis). This hyperventilation, in its extreme form, may be observed as Kussmaul respiration. DKA is common in type 1 diabetes as this form of diabetes is associated with an absolute lack of insulin production by the islets of Langerhans. In type 2 diabetes, insulin production is present but is insufficient to meet the body’s requirements as a result of end-organ insulin resistance. Usually, these amounts of insulin are sufficient to suppress ketogenesis. If DKA occurs in someone with type 2 diabetes, their condition is called “ketosis-prone type 2 diabetes”. The exact mechanism for this phenomenon is unclear, but there is evidence both of impaired insulin secretion and insulin action. Once the condition has been treated, insulin production resumes and often the patient may be able to resume diet or tablet treatment as normally recommended in type 2 diabetes.

Question 10

Three stages of DKA severity

Answer 10

Mild: blood pH mildly decreased to between 7.25 and 7.30 (normal 7.35–7.45); serum bicarbonate decreased to 15–18 mmol/l (normal above 20); the patient is alert. Moderate: pH 7.00–7.25, bicarbonate 10–15, mild drowsiness may be present. Severe: pH below 7.00, bicarbonate below 10, stupor or coma may occur

Question 11

symptoms of diabetes

Answer 11

Polyuria (urinating a lot), polydipsia (drinking a lot) and weight loss. Hemoglobin A1c ≥ 6.5%, Fasting plasma glucose ≥ 126 mg/dL, 2 hour plasma glucose ≥ 200 mg/dL, or Random plasma glucose ≥ 200 mg/dL in a patient with classic symptoms of hyperglycemia.

Question 12

Diabetic ketoacidosis

Answer 12

Hyperglycemia- Plasma glucose >200 mg/dL, Metabolic acidosis, Venous pH < 7.3 and/or HCO3- < 15 mmol/L. Ketonemia and ketonuria

Question 13

Ketonemia

Answer 13

the presence of ketones, mainly acetone, in the blood. It is characterized by the fruity breath odor of ketoacidosis.

Question 14

ketonuria

Answer 14

a medical condition in which ketone bodies are present in the urine. Ketones are metabolic end-products of fatty acid metabolism. In healthy individuals, ketones are formed in the liver and are completely metabolized so that only negligible amounts appear in the urine. However, when carbohydrates are unavailable or unable to be used as an energy source, fat becomes the predominant body fuel instead of carbohydrates and excessive amounts of ketones are formed as a metabolic byproduct. Higher levels of ketones in the urine indicate that the body is using fat as the major source of energy. Ketone bodies that commonly appear in the urine when fats are burned for energy are acetoacetate and beta-hydroxybutyric acid. Acetone is also produced and is expired by the lungs. Normally, the urine should not contain a noticeable concentration of ketones to give a positive reading. As with tests for glucose, acetone can be tested by a dipstick or by a lab. The results are reported as small, moderate, or large amounts of acetone. A small amount of acetone is a value under 20 mg/dl; a moderate amount is a value of 30–40 mg/dl, and a finding of 80 mg/dl or greater is reported as a large amount.

Question 15

normal venous blood pH and HCO3 levels

Answer 15

venous blood pH = 7.36

Question 16

normal arterial blood pH and HCO3 levels

Answer 16

arterial blood pH = 7.41

Question 17

beta pancrease cells

Answer 17

The primary function of a beta cell is to store and release insulin. Insulin is a hormone that brings about effects which reduce blood glucose concentration. Beta cells can respond quickly to spikes in blood glucose concentrations by secreting some of their stored insulin while simultaneously producing more. the only cells not working in the pancrease during diabetes

Question 18

Control of Insulin Secretion

Answer 18

Voltage gated calcium ion channels and ATP-sensitive potassium ion channels are embedded in the cell surface membrane of beta cells. These ATP-sensitive potassium ion channels are normally open and the calcium ion channels are normally closed. Potassium ions diffuse out of the cell, down their concentration gradient, making the inside of the cell more negative with respect to the outside (as potassium ions carry a positive charge). At rest, this creates a potential difference across the cell surface membrane of -70mV. When the glucose concentration outside the cell is high, glucose molecules move into the cell by facilitated diffusion, down its concentration gradient through the GLUT2 transporter. Since beta cells use glucokinase to catalyze the first step of glycolysis, metabolism only occurs around physiological blood glucose levels and above. Metabolism of the glucose produces ATP, which increases the ATP to ADP ratio. The ATP-sensitive potassium ion channels close when this ratio rises. This means that potassium ions can no longer diffuse out of the cell. As a result, the potential difference across the membrane becomes more positive (as potassium ions accumulate inside the cell). This change in potential difference opens the voltage-gated calcium channels, which allows calcium ions from outside the cell to diffuse in down their concentration gradient. When the calcium ions enter the cell, they cause vesicles containing insulin to move to, and fuse with, the cell surface membrane, releasing insulin by exocytosis.

Question 19

Insulin actions

Answer 19

can be summed up (and more easily remembered) by thinking of it as a signal to “lock up” energy. It stimulates uptake of glucose and triglycerides while promoting synthesis of fats, proteins, and glycogen. Liver: + glucose uptake, glycogen synthesis, - gluconeogenesis, - ketogenesis, + lipogenesis. Muscle: + glucose uptake, glycogen synthesis, + protein synthesis. Adipose, + glucose uptake, + triglyceride uptake, + lipid synthesis

Question 20

Insulin deficiency

Answer 20

Glucose cannot be taken into cells, despite adequate supply-> Hyperglycemia. The body needs another source of energy: Lipolysis à Fatty acid oxidation (liver) à Ketoacids (acetoacetate, betahydroxybutyrate)

Question 21

Compensation for the acidosis

Answer 21

acidosis drives the reaction: H+ + HCO3- H2CO3 H20 + CO2. Excess CO2 is eliminated through the lungs-> Kussmaul respirations

Question 22

Reason for dehydrantion in DKA

Answer 22

The dehydration seen in DKA is largely the result of the osmotic diuresis secondary to hyperglycemia. In the normal state, blood is filtered at the glomerulus, and as the filtrate courses through the nephron, the parsimonious kidney reabsorbs all the glucose filtered. After all, it is an energy source. However, in diabetes the blood glucose (and therefore filtrate glucose) concentration rises above levels which can be reabsorbed through facilitated diffusion (all the transporter proteins are working at capacity), and glucose is lost in the urine. With an excess of glucose (and therefore osmols) in the filtrate, the body is unable to reabsorb as much water as it would under normal conditions. Therefore, large volumes of water are lost in the urine, even in the setting of dehydration. Meanwhile, as the blood ketoacid concentration rises, the patient experiences nausea and vomiting, making it even more difficult for oral intake to keep up with urine volume loss, resulting in dehydration. Furthermore, dehydration leads to an even further concentration of blood glucose, worsening the pace of the osmotic diuresis, a spiral that can often only be reversed with IV hydration.

Question 23

Reasons for low K with DKA

Answer 23

As the patient fights dehydration, the body compensates by holding onto sodium more avidly. Increased sodium conservation in the distal convoluted tubule and cortical collecting duct of the nephron is mediated by aldosterone, which stimulates an antiport mechanism whereby sodium is retained at the expense of potassium loss in the urine. Therefore, patients in DKA are depleted in their total body potassium. Recall, however, that most of the body’s potassium is intracellular. Acidosis leads to influx of hydrogen ions into the cells, leading to a charge neutral efflux of potassium. Therefore, the acidosis can cause an excess of extracellular potassium (hyperkalemia) despite the overall depletion. As you begin treating this patient, the large doses of intravenous insulin which you will be providing lead to potassium shift intracellularly, lowering the blood potassium levels. Putting these processes together, you can see that a patient in DKA may present with hyperkalemia but will manifest greater potassium needs than most other patients you encounter. Therefore, as you treat this patient, you must continue to watch serum potassium levels and be prepared to make adjustments to the potassium content of IV fluids in order to prevent severe hyperkalemia and severe hypokalemia, both of which can lead to death. If this hasn’t sufficiently frightened you in to paying attention, note that potassium chloride is the “lethal” part of the three drug lethal injection cocktail used by the majority of the 32 states with the death penalty.

Question 24

How to treat DKA

Answer 24

Hyperglycemia: IV fluids (dilution) and Insulin Dehydration: Give Isotonic IV fluids Acidosis: Insulin (stop ketone formation), Fluids (urinary excretion of acid and ketones). Potassium: Total body K+ depletion and Needs lots of K+, but carefully

Question 25

Cerebral edema with DKA

Answer 25

occurring in about 0.15-0.3% of all cases of pediatric DKA, is the leading cause of morbidity and mortality, with a death rate ~24%. A further 20% of patients with cerebral edema suffer long term neurologic outcomes. The pathophysiology of cerebral edema is multifold and incompletely understood. Acidosis leads to dysregulated cerebral blood flow and perhaps even disruptions at the level of the blood brain barrier. Part of the process can be iatrogenic as we rehydrate the patient with relatively hypotonic fluids. Therefore, we aim to replace fluid losses more slowly than you would in cases of non-diabetic dehydration, preventing decreases in blood sodium concentration and slowing the rate of decrease of blood glucose. Overly rapid rehydration and hypotonic IV fluids can precipitate cerebral edema, which manifests as: mental status changes; headache; Cushing’s triad (hypertension, bradycardia, irregular respirations); or fixed, dilated pupils.

Question 26

Treatment for cerebral edema

Answer 26

Treatment for cerebral edema includes elevating the head of the bed, hyperventilating the patient (if intubated) [N.B. Rapid decrease in serum CO2 constricts cerebral arteries and therefore decreases cerebral blood flow], and giving IV mannitol or hypertonic saline. Mannitol is a sugar alcohol which is only slightly metabolized (if at all). Given via IV, it serves to raise the effective osmolality of the blood and pull water back from the brain in order to decrease swelling.

Question 27

risk factors for cerebral edema

Answer 27

More frequent in New onset (risk 3x established diabetes), Younger (<5 years), Longer duration of symptoms, More severe DKA (Higher BUN, More severe acidosis, Lower pCO2, Higher [K+]). Treatment factors: Lesser rise in [Na+] during treatment, Insulin within the first hour of fluid therapy, Bicarbonate administration, High volume of fluid in first 4 hrs

Question 28

Cushing’s triad

Answer 28

a physiological nervous system response to increased intracranial pressure (ICP) that results in Cushing’s triad of increased blood pressure, irregular breathing, and a reduction of the heart rate. It is usually seen in the terminal stages of acute head injury and may indicate imminent brain herniation. It can also be seen after the intravenous administration of epinephrine and similar drugs.

Question 29

acidosis with DKA

Answer 29

The acidosis seen in DKA is a result of beta oxidation of fatty acids. This process generates hydrogen ions and ketone bodies (acetoacetate and betahydroxybutyrate) which we can measure. To compensate for the excess acid, the body increases respiratory volume and rate to hasten the elimination of carbon dioxide, taking advantage of the equation:H+ + HCO3- H2CO3 H20 + CO2. Recall that CO2 is very membrane permeable and readily removed from the body at a rate dependent on the volume of air ventilated per unit of time (usually discussed as “minute ventilation,” and dependent on respiratory rate and the volume of air exhaled with each breath). The deep and rapid respirations seen in DKA are “Kussmaul respirations.” Easy to remember: K for Kussmaul, K for ketoacidosis.

Question 30

intravenous insulin

Answer 30

The intravenous insulin serves two purposes: decreasing the blood glucose concentration and halting ketoacid production. However, it invariably takes a longer time to resolve the ketoacidosis than to decrease the glucose to normal levels. Therefore, you will paradoxically need to start an intravenous infusion of dextrose hours before stopping the intravenous insulin. Two cardinal sins in DKA management are prematurely stopping the insulin infusion and failing to use enough dextrose to bring the blood glucose slowly into the target range.

Question 31

Insulin

Answer 31

Insulin is a 51 amino acid protein released by the beta cells of the pancreas. In type 1 diabetes mellitus, an incompletely understood autoimmune process leads to destruction of these beta cells and results in insulin deficiency. Insulin release is regulated by a glucose sensing system within the beta cells: glucose enters the cell through the GLUT2 transporter, undergoes glycolysis, leading to an increase in the intracellular ATP to ADP ratio. This change closes the ATP-sensitive potassium channel, preventing outward leak of potassium ions. The resultant buildup of intracellular potassium (because there are other sodium/potassium channels actively antiporting against the gradient) depolarizes the membrane, activating a voltage-gated calcium channel and leading to calcium influx. The increased intracellular calcium ion concentration leads to exocytosis of preformed insulin-containing secretory granules. Membrane proteins in this system, including some I did not discuss here, are potential drug target sites and can be deranged in some forms of monogenic diabetes and hyperinsulinism. Once released from the beta cell into the bloodstream, human insulin has a half-life of about 5 minutes, allowing for minute-by-minute fine control of blood sugar.

Question 32

stimulus for insulin release

Answer 32

Glucose enters the beta cells in the pancreas à increased ATP/ADP ratio à closes a channel to cause rising intracellular potassium à increasing intracellular potassium depolarizes the membrane à Calcium ions influx à leads to insulin exocytosis

Question 33

Li Fraumeni like syndrom

Answer 33

only 40% are p53 related. he Birch definition still requires three affected individuals in the family and the Eeles definition used slightly looser criteria to define the so-called Li-Fraumeni-like syndrome in patients who do not meet the classical Li-Fraumeni syndrome criteria. These Li-Fraumeni-like syndrome definitions are also based upon the occurrence of typical malignancy in both the proband and one or more relatives at a relatively early age

Question 34

treatment of li fraumeni related cancer

Answer 34

The management of the various cancers is generally the same as that in patients without Li-Fraumeni syndrome. However, for women with breast cancer, mastectomy rather than lumpectomy followed by radiation therapy is generally preferred because of the risks of second breast cancers or radiation-induced neoplasms

Question 35

diagnostic criteria of LFS

Answer 35

The original descriptions of Li-Fraumeni syndrome served as the basis for a clinical diagnosis, which was defined as follows: A proband with a sarcoma diagnosed before age 45 years AND, A first-degree relative with any cancer before age 45 years AND, A first- or second-degree relative with any cancer before age 45 years or a sarcoma at any age

Question 36

HER2

Answer 36

a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or overexpression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. In recent years the protein has become an important biomarker and target of therapy for approx. 30% of breast cancer patients. The ErbB family is composed of four plasma membrane-bound receptor tyrosine kinases, the other members being epidermal growth factor receptor, erbB-3 (neuregulin-binding; lacks kinase domain), and erbB-4.All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. HER2 can heterodimerise with any of the other three receptors and is considered to be the preferred dimerisation partner of the other ErbB receptors.

Question 37

Ras-Raf-MEK-ERK pathway

Answer 37

a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. Overall, the extracellular mitogen binds to the membrane receptor. This allows Ras (a GTPase) to swap its GDP for a GTP. It can now activate MAP3K (e.g., Raf), which activates MAP2K, which activates MAPK. MAPK can now activate a transcription factor, such as myc. Receptor-linked tyrosine kinases such as the epidermal growth factor receptor (EGFR) are activated by extracellular ligands. Binding of epidermal growth factor (EGF) to the EGFR activates the tyrosine kinase activity of the cytoplasmic domain of the receptor. The EGFR becomes phosphorylated on tyrosine residues. Docking proteins such as GRB2 contain an SH2 domain that binds to the phosphotyrosine residues of the activated receptor. GRB2 binds to the guanine nucleotide exchange factor SOS by way of the two SH3 domains of GRB2. When the GRB2-SOS complex docks to phosphorylated EGFR, SOS becomes activated. Activated SOS then promotes the removal of GDP from a member of the Ras subfamily (most notably H-Ras or K-Ras). Ras can then bind GTP and become active. In many cancers (e.g. melanoma), a defect in the MAP/ERK pathway leads to that uncontrolled growth. Many compounds can inhibit steps in the MAP/ERK pathway, and therefore are potential drugs for treating cancer, e.g., Hodgkin disease. RAF-ERK pathway is also involved in the pathophysiology of Noonan’s Syndrome, a polymalformative disease, where Simvastatin was proposed as a way to improve CNS-cognitive symptoms of the disorder.

Question 38

myc pathway

Answer 38

Myc protein is a transcription factor that activates expression of many genes through binding on consensus sequences (Enhancer Box sequences (E-boxes)) and recruiting histone acetyltransferases (HATs). It can also act as a transcriptional repressor. By binding Miz-1 transcription factor and displacing the p300 co-activator, it inhibits expression of Miz-1 target genes. In addition, myc has a direct role in the control of DNA replication. Myc is activated upon various mitogenic signals such as Wnt, Shh and EGF (via the MAPK/ERK pathway). By modifying the expression of its target genes, Myc activation results in numerous biological effects. The first to be discovered was its capability to drive cell proliferation (upregulates cyclins, downregulates p21), but it also plays a very important role in regulating cell growth (upregulates ribosomal RNA and proteins), apoptosis (downregulates Bcl-2), differentiation, and stem cell self-renewal. Myc is a very strong proto-oncogene and it is very often found to be upregulated in many types of cancers. Myc overexpression stimulates gene amplification, presumably through DNA over-replication. c-Myc induces AEG-1 or MTDH gene expression and in turn itself requires AEG-1 oncogene for its expression.

Question 39

cdkn2a

Answer 39

p16 (also known as cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 and as several other synonyms), is a tumor suppressor protein, that in humans is encoded by the CDKN2A gene. epigenetic silencing by methylation can lead to cancer. p16 plays an important role in cell cycle regulation by decelerating cells progression from G1 phase to S phase, and therefore acts as a tumor suppressor that is implicated in the prevention of cancers, notably melanoma, oropharyngeal squamous cell carcinoma, and esophageal cancer. The CDKN2A gene is frequently mutated or deleted in a wide variety of tumors. p16 is a cyclin-dependent kinase (CDK) inhibitor that slows down the cell cycle by prohibiting progression from G1 phase to S phase. Normally, CDK4/6 binds cyclin D and forms an active protein complex that phosphorylates retinoblastoma protein (pRB). Once phosphorylated, pRB disassociates from the transcription factor E2F1, liberating E2F1 from its cytoplasm bound state allowing it to enter the nucleus. Once in the nucleus, E2F1 promotes the transcription of target genes that are essential for transition from G1 to S phase. p16 acts as a tumor suppressor by binding to CDK4/6 and preventing its interaction with cyclin D. This interaction ultimately inhibits the downstream activities of transcription factors, such as E2F1, and arrests cell proliferation. This pathway connects the processes of tumor oncogenesis and senescence, fixing them on opposite ends of a spectrum. On one end, the hypermethylation, mutation, or deletion of p16 leads to downregulation of the gene and can lead to cancer through the dysregulation of cell cycle progression. Conversely, activation of p16 through the ROS pathway, DNA damage, or senescence leads to the buildup of p16 in tissues and is implicated in aging of cells.

Question 40

function of p53

Answer 40

p53 has many mechanisms of anticancer function, and plays a role in apoptosis, genomic stability, and inhibition of angiogenesis. In its anti-cancer role, p5 3 works through several mechanisms: It can activate DNA repair proteins when DNA has sustained damage. Thus, it may be an important factor in aging. It can arrest growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle). It can initiate apoptosis - programmed cell death - if DNA damage proves to be irreparable. Activated p53 binds DNA and activates expression of several genes including microRNA miR-34a, WAF1/CIP1 encoding for p21 and hundreds of other down-stream genes. p21 (WAF1) binds to the G1-S/CDK (CDK4/CDK6, CDK2, and CDK1) complexes (molecules important for the G1/S transition in the cell cycle) inhibiting their activity. When p21(WAF1) is complexed with CDK2 the cell cannot continue to the next stage of cell division. A mutant p53 will no longer bind DNA in an effective way, and, as a consequence, the p21 protein will not be available to act as the “stop signal” for cell division.[29] Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this cell type, p53 activates numerous microRNAs (like miR-302a, miR-302b, miR-302c, and miR-302d) that directly inhibit the p21 expression in hESCs.

Question 41

regulation of p53

Answer 41

p53 becomes activated in response to myriad stressors, including but not limited to DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress,[35] osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. This activation is marked by two major events. First, the half-life of the p53 protein is increased drastically, leading to a quick accumulation of p53 in stressed cells. Second, a conformational change forces p53 to be activated as a transcription regulator in these cells. The critical event leading to the activation of p53 is the phosphorylation of its N-terminal domain. The N-terminal transcriptional activation domain contains a large number of phosphorylation sites and can be considered as the primary target for protein kinases transducing stress signals. The protein kinases that are known to target this transcriptional activation domain of p53 can be roughly divided into two groups. A first group of protein kinases belongs to the MAPK family (JNK1-3, ERK1-2, p38 MAPK), which is known to respond to several types of stress, such as membrane damage, oxidative stress, osmotic shock, heat shock, etc. A second group of protein kinases (ATR, ATM, CHK1 and CHK2, DNA-PK, CAK, TP53RK) is implicated in the genome integrity checkpoint, a molecular cascade that detects and responds to several forms of DNA damage caused by genotoxic stress. Oncogenes also stimulate p53 activation, mediated by the protein p14ARF. In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 (also called HDM2 in humans), which is itself a product of p53, binds to p53, preventing its action and transports it from the nucleus to the cytosol. Also Mdm2 acts as ubiquitin ligase and covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome. However, ubiquitylation of p53 is reversible.

Question 42

p53’s role in disease

Answer 42

If the TP53 gene is damaged, tumor suppression is severely compromised. People who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disorder known as Li-Fraumeni syndrome. The TP53 gene can also be modified by mutagens (chemicals, radiation, or viruses), increasing the likelihood for uncontrolled cell division. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Increasing the amount of p53 may seem a solution for treatment of tumors or prevention of their spreading. This, however, is not a usable method of treatment, since it can cause premature aging. Restoring endogenous normal p53 function holds some promise. Research has showed that this restoration can lead to regression of certain cancer cells without damaging other cells in the process. The ways by which tumor regression occurs depends mainly on the tumor type. For example, restoration of endogenous p53 function in lymphomas may induce apoptosis, while cell growth may be reduced to normal levels. Thus, pharmacological reactivation of p53 presents itself as a viable cancer treatment option. Loss of p53 creates genomic instability that most often results in an aneuploidy phenotype. Certain pathogens can also affect the p53 protein that the TP53 gene expresses. One such example, human papillomavirus (HPV), encodes a protein, E6, which binds to the p53 protein and inactivates it. This mechanism, in synergy with the inactivation of the cell cycle regulator pRb, by the HPV protein E7, allows for repeated cell division manifested clinically as warts. Certain HPV types, in particular types 16 and 18, can also lead to progression from a benign wart to low or high-grade cervical dysplasia, which are reversible forms of precancerous lesions. Persistent infection of the cervix over the years can cause irreversible changes leading to carcinoma in situ and eventually invasive cervical cancer. This results from the effects of HPV genes, particularly those encoding E6 and E7, which are the two viral oncoproteins that are preferentially retained and expressed in cervical cancers by integration of the viral DNA into the host genome. The p53 protein is continually produced and degraded in cells of healthy people. The degradation of the p53 protein is associated with binding of MDM2. In a negative feedback loop, MDM2 itself is induced by the p53 protein. Mutant p53 proteins often fail to induce MDM2, causing p53 to accumulate at very high levels. Moreover, the mutant p53 protein itself can inhibit normal p53 protein levels. In some cases, single missense mutations in p53 have been shown to disrupt p53 stability and function.

Question 43

structure of p53

Answer 43

Has the following domains 1) an acidic N-terminus transcription-activation domain (TAD), also known as activation domain 1 (AD1), which activates transcription factors 2) activation domain 2 (AD2) important for apoptotic activity. 3) Proline rich domain important for the apoptotic activity of p53. 4) central DNA-binding core domain (DBD). Contains one zinc atom and several arginine amino acids. 5) nuclear localization signaling domain. 6) homo-oligomerisation domain (OD): residues 307-355. Tetramerization is essential for the activity of p53 in vivo. 7) C-terminal involved in downregulation of DNA binding of the central domain. Mutations that deactivate p53 in cancer usually occur in the DBD. 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. As such, mutations in the DBD are recessive loss-of-function mutations. Molecules of p53 with mutations in the OD dimerise with wild-type p53, and prevent them from activating transcription. Therefore OD mutations have a dominant negative effect on the function of p53.

Question 44

p21

Answer 44

p21 is a potent cyclin-dependent kinase inhibitor (CKI). The p21 (CIP1/WAF1) protein binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a regulator of cell cycle progression at G1 and S phase. In addition to growth arrest, p21 can mediate cellular senescence. One of the ways it was discovered was as a senescent cell-derived inhibitor. The expression of this gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. p21 can also interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation. However p21 may inhibit apoptosis and does not induce cell death on its own. Two alternatively spliced variants, which encode an identical protein, have been reported. Sometimes p21 is expressed without being induced by p53. This kind of induction plays a big role in p53 independent differentiation which is promoted by p21. Expression of p21 is mainly dependent on two factors 1) stimulus provided 2) type of the cell. Growth arrest by p21 can promote cellular differentiation. p21 therefore prevents cell proliferation. Despite regulation by tumor suppressor gene p53, loss-of-function mutations in p21 (unlike p53) do not accumulate in cancer nor do they predispose to cancer incidence. Mice genetically engineered to lack p21 develop normally and are not susceptible to cancer at a higher rate than wild-type mice (unlike p53 knockout mice).