Distinguish between karyokinesis and cytokinesis and relate each to the stages of mitosis.

Mitosis is made of:

Karyokinesis (nuclear division) (PMAT) &
Cytokinesis (cytoplasm division) (happens at the end of Telophase)

Discuss the changes that the nuclear membrane undergoes during cell division.

Prophase: Nucleolus disappears, Nuclear envelope disappears as proteins of the nuclear membrane and lamina are phosphorylated

Anaphase: Reconstitution of nuclear 
envelope around chromosomes, nucleolus reapears

Compare and contrast mitosis and meiosis.

Mitosis: 1 replication of DNA, 1 division; 2 identical diploid cells (2n) produced;

Meiosis: 1 replication of DNA, 2 divisions, 4 genitically unique haploid cells (n) produced

Identify the stages of the cell cycle and describe the events that occur in each stage. Place these events in context of mitosis.

Cell Cycle - IPMAT; PMAT is mitosis and I is interphase

G1 (first gap) - Synthesis of RNA and proteins
Nucleotides and enzymes needed for division accumulate; Lasts a few hours to life of cell (25 hours)

S (DNA replication) - Replication of DNA and histones occurs; Centrioles duplicate (Lasts ~ 7 hours); Cells in this phase can be labeled with tritiated thymidine or bromodeoxyuridine

G2 (second gap) - No DNA synthesis, Last 1 hour to life of cell (4-5 hours)

Describe the process by which dynamic changes in cyclin and cyclin-dependent kinases regulate cell cycle progression.

Progression of cells through (each stage of) cell cycle is controlled by cyclin/cdk complexes. Composed of:

• Cyclins–regulatory subunit
• Cyclin-dependent kinases (Cdks)–catalytic subunit that regulates cell cycle by phosphorylating critical proteins.

Both must be present for the complex to be active; cdk leves constant, but cyclin levels can fluctuate. Its levels are regulated by degradation and inhibition:

• ubiquitin is added to cyclin targeting it for degradation by proteosomes–a large protein complex that degrades proteins tagged with ubiquitin 

• cdk inhibitors can bind to the cyclin-cdk complex and inhibit its activity. Examples: p27 and p21

Discuss cell cycle check points with respect to the cellular events that are monitored, the molecules that control cell cycle progression, and the potential consequences of failure to pass the checkpoint.

G1 - DNA-damage checkpoint–high levels of p53 (a tumor suppressor) are triggered by DNA damage and preclude entry into S phase. (p53 defects/loss are seen in half of all malignancies)

G1 - Restriction checkpoint (point of no return) –cells physiological properties are checked–nutrients, cell size, matrix attachment. Mediated by interactions between:
• E2F a family of transcription factors required for expression of molecules required for cell cycle progression (including cyclins and cdks) 
• Rb (Retinoblastoma susceptibility protein) binds to and inhibits E2F. Loss or inactivation of RB results in malignancy.

S phase DNA-damage checkpoint–monitors quality of replicating DNA 

G2 phase DNA-damage checkpoint 
G2 Unreplicated DNA checkpoint–prevents progression to mitosis until DNA synthesis is complete. 

Mitosis (M phase) - Spindle assembly checkpoint–detects failure of spindle fibers to attach to kinetochores; arrest cells in metaphase; Detects improper alignment of spindle and blocks cytokinesis. Triggers apoptosis if damage is irreparable.

ANY fail at DNA damage checkpoints causes...

Mitotic catastrophe–failure to arrest the cell cycle before mitiosis resulting in aberrant chromosome segregation

Fail at (M) Spindle assembly checkpoint causes aneuploidy–cells with aberrant chromosome number

Fail at (G1) Restriction checkpoint causes...

cells to lose contact inhibition–cessation of cell division when cells are ate high density

Stem cell populations are in G₀ and can be re-activated to enter cycle in response to injury of body tissues by cytokines.

How is cellular self-renewal status characterized and what are some means for measuring the self-renewal status of cells.

• Self renewal–the maintenance of the stem cell population due to the fact that some of the daughter cells remain undifferentiated following cell division. Other daughter cells differentiate.

Type of population:

• Stable populations–cells divide occasionally to maintain normal tissue or organ structure
• terminally differentiated—cells that cannot re-enter

cell cycle and exist briefly. Examples: some cells of the blood and the epidermis. Progenitors that rapidly divide produce these cells as they transit from stem cells to terminally differentiated cells.

Speed of renewal:

•Renewing cell populations–display regular mitotic activity

• can be slowly self renewing (smooth muscle of hollow organs  
• or rapidly self-renewing dividing (blood and epithelial cells)

Measurement of renewal

1. level of mitotic activity, stimated by the #of mitotic metaphases seen in a single high magnification field

2. Flow cytometry assesses: (1) Cell cycle, (2) Expression of molecules associated with proliferation, (3) Loss of membrane tracking dyes.

Compare and contrast physiological and pathological cell death.

•Apoptosis– normal programmed cell death, regulates number of cells in tissues such as epidermis, GI tract,
hematopoietic system; necessary for survival of organism.

•Necrosis–death due to irreversible (pathologic) exogenous injury; unregulated & harmful to the organism

Describe cellular features that are characteristic of necrosis and of apoptosis and how these two mechanisms of cell death can be distinguished.

Apoptosis

• Cell shrinkage–entire cell contents condense: DNA fragmentation and condensation (pyknosis)
• Membrane blebbing–release of cell contents in
membrane-enclosed apoptotic bodies. 
• Contents are not released extracellularly
• No inflammatory response, but phagocytosis of apoptotic cells or bodies.
• Involves individual cells or small groups of cells;
necrosis involves large areas of cell death

Necrosis

•cell swelling due to loss of plasma membrane integrity and ion pump function
•organelles such as mitochondria and rER undergo irreversible changes caused by swelling
•intracellular contents, including lysosomal enzymes are spilled into extracellular space resulting in surrounding tissue damage
•released cell contents stimulate the immune system producing an intense inflammatory response

Discuss the cellular and biochemical features of apoptosis and its physiologic role.

Biochemistry of apoptosis:

• Activation of caspases–a cascade of proteolytic enzymes in the cytoplasm.   
• DNA breakdown into 50 to 300 kb pieces
• DNA is cleaved b/w histones producing fragments of 180-200 bp
• phosphatidylserine translocated from the inner to the outer layer of bilayer of the plasma membrane. This targets the cell for apoptosis which can occur before they reach later stages of apoptosis.
• Depolarization of mitochondrial membrane:
cytochrome c from the mitochondrial matrix is released into the cytoplasm where it can activate caspases.

Physiology of apoptosis:

• cell deletion in proliferating cell populations  
• death of cells which have served their purpose  
• neutrophils in inflammation, lymphocytes after immune response, elimination of self-reactive lymphocytes
• cell death induced by cytotoxic T cells (eliminates virus-infected and neoplastic cells)
• If DNA defects due to the following cannot be repaired, the cell is induced to undergo apoptosis:   

 
1. infidelity of DNA polymerase
2. UV light, ionizing radiation
3. DNA binding chemicals

• viral infections, however, some viral gene products have ability to inhibit apoptosis

Compare the intrinsic and extrinsic pathways of apoptosis with respect to initiating events, mechanisms of regulation, and convergence. 

Extrinsic pathway–death receptor initiated 
• Ligands are produced by other cells and
stimulate death receptors on target cells. Examples: 
 –fas ligand (found on membrane of immune cells) binds to Fas (CD95)     
 –soluble tumor necrosis factor (TNF) binds TNF receptor
• These transmembrane receptors have amino
acid sequences called “death domains” that
bind to death domains of docking proteins
(such as FADD) resulting in caspase
activation

intrinsic pathway–mitochondrial 
• Triggered by cellular stress (oxidative and DNA damage) resulting in increased mitochondrial permeability and release of pro-apoptotic molecules into the cytoplasm (Bax, Bim, Bak, Bik, Bad)

• Bcl-2 family proteins are found in the inner mitochondrial membrane and are either pro- or anti-apoptotic: (Bcl-2, Bcl-XL); Loss of them allows proteins such as cytochrome C to leak out of the mitochondrion and activate the caspase cascade

• ROS (reactive oxygen species) such as peroxide stimulate mitochondria to release cytochrome C
• p53 binds to damaged areas of DNA; activates proteins which arrest G1, allowing time to direct repair enzymes for DNA repair; If repair isn’t possible, it activates apoptotic pathway: activates apoptosis by increasing production of Bax and Fas, and decreasing Bcl-2, and translocating Fas (extrinsic factor) from membrane to cytosol

Identify assays that are used to measure apoptosis and describe how they measure the biological events that are characteristic of apoptosis.