Episode 54: Learn about the spectrin defect causing spherocytosis, the ubiquitination of damages intracellular constituents, different types of cells, regulation of the cell cycle, and cancer drugs.

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I. Cell Membrane Defects

A. RBC membrane defect:

Spherocytosis is a defect in spectrin within RBC cell membrane; if you can’t see a central area of pallor (if you don’t see a donut) then it’s a spherocyte. Absence of spectrin with in the RBC does not allow the RBC to form a biconcave disk; it is defective, and therefore forms a sphere.

B. Ubiquitin

stress protein. High ubiquitin levels are associated with high levels of stress.

Some of the intermediate filaments (keratin, desmin, vimentin) are part of the superstructure of our cells (“frame of the cell”, upon which things are built). When these intermediate filaments get damaged, the ubiquitin marks then for destruction. The intermediate filaments have been tagged (ubiquinated) and marked for destruction. Some of these products have names, for example: there are open spaces within the liver tisse, these spaces are fat and they are probably due to alcohol. The ubiquinited products of the liver are called Mallory bodies. These are the result of ubiquinated filaments called keratin and these are seen in alcoholic hepatitis. Another example: Silver stain of neurofibilary tangles – Jacob crutzfelt and alzheimers dz. Tau protein is associated with neurofib tangles; this is an example of a ubiquinated neurofilament.

Example: Substantia nigra in Parkinson’s Dz – include inclusions called Lewy bodies, neurotransmitter deficiency is dopamine. Lewy bodies are ubiquinated neurofilaments. Therefore, Mallory bodies, Lewy bodies, and neurofib tangles are all examples of ubiquintation.

II. Cell Cycle

A. Different types of cells:

1. Labile cells

cell where the division is via a stem cell. Three tissues that has stem cells: bone marrow, basement membrane of skin, and the base of crypts in the intestine. These cells have the tendency of being in the cell cycle a lot. In pharm: there are cell cycle specific and cell cycle nonspecific drugs. The cells that are most affected by these drugs are the labile cells b/c they are in the cell cycle. Complications of these drugs are BM suppression, diarrhea, mucocidis, and rashes on the skin (there are stem cells in all these tissues!).

2. Stable cells

in resting phase, Go phase. Most of parenchymal organs (liver, spleen, and kidney) and smooth muscle are stable cells. Stable cells can ungo division, but most of the time they are resting, and something must stimulate them to get into the cell cycle and divide – ie a hormone or a growth factor. For example: estrogen in woman will help in the proliferative phase of the menstrual cycle. The endometrial cells are initially in the Go phase and then the estrogen stimulated the cells to go into the the cell cycle. Therefore, they can divide, but they have to be invited by a hormone or a growth factor.

3. Permanent cells

can no longer get into the cell cycle, and have been permanently differentiated. The other types of muscle cells: striated, cardiac and neuronal cells. Only muscle that is NOT a permanent tissue = smooth muscle; hyperplasia = increase in #, while hypertrophy = increase in size. Would a permanent cell be able to under hyperplasia? NO, b/c that means more copies of it. Can it go under hypertrophy? Yes. A smooth muscle cell can undergo hyperplasia AND hypertrophy.

B. Different phases of cell cycle:

1. G1 phase:

The most variable phase of cell cycle is the G1 phase. Compare with menstrual cycle: The most variable phase is the proliferative phase (not the secretory phase). The prolifertive phase varies with stress; however, once ovulation has occurred, it is 14 days. Therefore, proliferative phase is analogous to G1 phase of the cell cycle b/c it can be shorter or lengthened; none of the other phases (S, G2, and M phase) changes, they stay the same. Therefore, in cancer cells, ones with a longer cell cycle will have a longer G1 phase, and cancer cells with a shorter cell cycle will have a shorter G1 phase.

G1 phase is the mastermind of everything. Cyclin dependent kinase (kinase = phosphorylation = activation). Phosphorylation usually involves sending a message to activate something. Glucagon is a  phosphorylator, while insulin is a dephosphorylator. Glucagon will phosphorylate protein kinase and activate it, while Insulin would dephosphorylate protein kinase and inactivate it.

G1 to S phase:

Inactive Cyclin d dependent kinase: Cyclin d activates it, and G1 phase makes cyclin D. Once cyclin D is made in the G1 phase, it then activates the enzyme: cyclin dep. kinase (therefore it is now active). Key area to control in cell cycle: transition from G1 to S phase. Because if you have a mutation and it goes into S phase, it then becomes duplicated, then you have the potential for cancer. Two suppressor genes that control the transition: (1) Rb suppressor gene: located on chromosome 13, which makes the Rb protein, which prevents the cell from going from the G1 to the S phase. In general, to go from G1 to S, the active cyclin dep kinase phosphorylates the Rb protein; when it is phosphorylated=activation, it can go from the G1 phase to the S phase. A problem occurs if there is a mutation. Therefore the enzyme is checked by (2) p53 suppressor gene: located on chromosome 17, which makes a protein product that inhibits the cyclin d dep kinase. Therefore, it cannot go into the S phase; p53 is the number 1, most imp gene that regulates human cancer.

Example: HPV – inactivates Rb suppressor gene and p53 suppressor gene. HPV makes two genes products – E6 (which knocks off the p53) and E7 (which knocks of the Rb suppressor gene).

If you have a point mutation the Rb suppressor gene, the Rb suppressor gene is knocked off, there will be no Rb protein, and the cell will progress to the S phase b/c it is uncontrolled. This mutation in the Rb suppressor gene predisposing to many cancers, such as retinoblastoma, osteogenic sarcoma (ie kid with pain around knees, Codman’s triangle – sunburst appearance on x-rays), and breast cancer (Rb suppressor can be involved). Depending on the age bracket, it hits in different areas. If you knock of p53 suppressor gene: the kinase will be always active, it will always phosphorylate the Rb protein, and that means that it will always go into the S phase, and this is bad. If you knock off any of those genes, the cell will go into the S phase. The p53 suppressor gene is the guardian of the genome, b/c it gives the cell time to detect if there are any defects/abnormalities in the DNA (splicing defects, codon thing, whatever, etc). DNA repair enzymes can splice out the abnormality, correct it, and the cell is ready to go to the S phase. If the cell has too much damaged DNA, then it is removed by apoptosis. Therefore this gene is imp b/c it gives the cell an opportunity to clean its DNA before going into the S phase.

2. S phase =

synthesis phase, where everything is doubled, includes DNA and chromosomes (from 2N to 4N). For example: if it’s in muscle, it will have double the number of contractile elements.

3. G2 phase =

where tubulin is made (imp to microtubule of the mitotic spindle); it is blocked by etoposide and bleomycin.

4. M phase =

mitosis; where the cell divides into two 2N cells. The cell can either go into the Go resting phase, or can continue dividing in the cycle, or can be permanently differentiated. p53 gene makes a protein to inhibit the kinase, therefore prevents the Rb protein from being phosphorylated, therefore stays in the G1 phase. Therefore, when you knock it off, no one is inactivating the kinase, and the cell is constantly phosphorylated and that keeps the cell dividing, and then has the potential to lead to cancer.

C. Drugs that act on the cell cycle:

1. Drugs acting on S phase:

a) Ergot alkaloids work on the mitotic spindle in S phase

b) Methotrexate works in S phase: Example: pt with rheumatoid arthiritis has macrocytic anemia. Drug responsible for this is in what phase of the cell cycle? S phase b/c it is methotrexate blocking dihydrofolate reductase

2. Drugs acting on G2 phase:

a) Etoposide

b) Bleomycin

3. Drugs acting on M phase:

a) Gresiofulvin in M phase

b) Paclitaxel specifically works in the M phase: Clinical scenario: this drug is a chemotherapy agent made from a yew tree? Paclitaxel (m phase)

c) Vincristine and Vinblastine

d) This drug used to be used for the treatment of acute gouty arthritis but b/c of all the side effects is no longer used. What drug and where does it act? Colchicine (m phase)

4. Clinical scenario:  A patient is on a drug that does not work on the cell cycle. There is a history of  HIV and complains of  dyspnea. There is white out of the lung on Chest x-ray. The patient deteriorates to cyanosis. Which drug? Dapsone

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