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Flashcards in LEC49: Intro to Cell Cycle Deck (45)
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techniques for measuring cell proliferation?

1) mitotic index 

2) Ki-67 antigen detection

3) flow cytometry


purpose of creating mitotic index?

quantitate the numebr of cells undergoing cell division


why/how does mitotic index count work?

chromsomes condense during mitosis, visualizable in metaphase

thus easy to see condensed chromosomes in light microscope during metaphase

can count the number of mitoses seen for a number of cells, = mitotic index 



mitotic index for proliferating tissues?



calculation of mitotic index?

mitotic index = # of mitotic cells / total # of cells


what is this count 

mitotic index 

see 3 cells undergoing mitosis, cell division


purpose of Ki-67 antigen detection?

detect proliferating cells

by immunohistochemistry


where is Ki-67 expressed?

in cells undergoing active division 

detectable using an antibody


what kind of tissue can be used for Ki-67 staining?

tissue section does not have to be fresh tissue 



what is this


ovarian cancer tissue section 

cancer cells are undergoing uncontrolled proliferation; distinguishable from surrounding normal cells due to brown stain for Ki-67 antigen expressed in proliferating cells


what does flow cytometry measure

flourescence per cell, thus measures DNA content


how does flow cytometry to measure DNA content work?

cells are incubated w/ a fluorescent dye, propidium iodide 

propidium iodide intercalates into genomic DNA 

single cell suspension is run through the flow cytometer 

laser shines a light w/ a specific wavelength on each individual cell 

detector measures fluorescence at distinct wavelength 

number of ells w/ each amt of fluorescence is then quanitated


in flow cytometry, what is wavelength of cells at different moments in the cell cycle?

G1 cells: 1x fluorescence 

G2, M cells: 2x fluorescence 

S phase: 1-2X fluorescence 

represents the relative amount of DNA each phase of cells has



what is flow cytometry often used to quantify?

anything that fluoresces 

often, a cell surface marker, i.e. a different kind of T cell, can determine which kind of T cell it is by flow cytometer b/c antibody recognizes a particular antigen and then do flourescence analysis 

often for blood analysis


structure of kinases?

a cyclin-cyclin-dependent complex of a cyclin and CDK

regulatory subunit: cyclin 

catalytic component: CDK, cyclin-dependent kinase

kinase needs cyclin to be active, function


what upregulates cyclin activity? 

what downregulates cyclin activity?

upregulation of cyclin: at transcription level 

downregulation of cyclin: at protein degredation level


CDK activity?

phosphorylate a target protein on either serine or thronine that immediately preced a proline residue

this phosphorylation event causes movement through the cell cycle


what are the phases of the cell cycle?

S, G2, M, G1


what happens during S phase?

DNA synthesis: 

DNA polymerase, ntds are needed for DNA polymerase to make more DNA


what happens during M phase?


chromosomal condensation 

nuclear membrane breaks down 

sister chromatids separate 

cytokinesis occurs 

occurs through phosphorylation events


what is the cyclin-CDK complex governing early G1?

Early G1: Cdk4/6, cyclin D 



what is the cyclin-CDK complex governing late G1?

Cdk2, Cycline E


what is the cyclin-CDK complex governing S phase?

Cdk2, Cyclin A


what is the cyclin-CDK complex governing G2-M transition?

Cdk1, Cyclin A & Cdk1, Cyclin B


how does a cyclin give activity to the CDK?

the substrate binding site spans both the cyclin and the CDK; substrate specificity is conferred by both the cyclin and the CDK 

know this b/c one kinase (Cdk2) can associate w/ 2 different cyclins at diff points in the cell cycle 


what does binding of the cyclin to the Cdk do?

1) provides part of the substrate binding site 

2) induces a conformational change that allows the substrate to access the catalytic site

cyclin binding is necessary but not sufficienct for Cdk activation


what determines cell cycle activity, besides cyclin-Cdk regulation?

1) cyclin levels 

2) phosphorylation of the CDK itself - activating & inhibiting phosphorylation 

3) inhibitors


is cyclin binding necessary/sufficient for Cdk activation?

cycling binding is necessary but not sufficient for Cdk activation



what must occur besides cyclin binding for activation of the Cdk?

describe the process

phophorylation of the Cdk subunit by CAK, Cdk Activating Kinase 

phosphorylates the catalytic subunit of all cyclin-dependent kinases on a conserved threonine residue (Thr 160) 

this phosphorylation is required for full activation of Cdks



describe the process of activating phosphorylation

when CAK phoshporylates the CDK on T160 site, this activates the kinase. process: 

before cyclin binding, part of the Cdk, the t-loop, sites in substrate binding site & prevents substrate binding 

binding of the Cyclin causes T-loop to shift, partially exposing substrate binding site

phoshporylation of the T-loop on T160 by CAK fully move the T-loop 

substrate binding site is thus completely exposed




what confers inhibitory phosphates

Wee1 enzyme


describe process of inhbitory phosphorylation and how it is counteracted

Wee1 kinase (enzyme) phosphorylates Cdks at T14 and T15 sites 

these residues are adjacent to ATP binding pocket; negative charges of added phosphates interferes w/ ATP binding, which is also negatively charged 

Cdc25 phosphatases must remove the phosphates to make Cyclin-CDK complex active



what counteracts inhibitory phosphorylation activity on Cyclin-CDK complexes? 

Cdc25 family phosphatases: Cdc25A, B C



describe interaction of positive feedback with Cyclin-CDK phosphorylation activity?

both the activating CAK phosphorylation and Wee1 inhibitory phosphorylation on kinase occur simultaneously

activating CAK phosphate cannot bind ATP when inhibitory Wee1 phosphate is present 

Cdc25 removes inhibitory phosphate, making an active complex 

positive feedback from that activated M-Cdk phosphorylates Cdc25 phosphatases and makes them more active to remove inhbitory phosphates; Cdks also phosphorylate Wee1 and prevent it from adding inhibitory phosphates

thus some amount of activated kinase now facilitates a lot more of the kinase becoming active, further enhances activity



classes of protein inhibitors of CDKs?

1) CIP family 

2) INK4 family


what are CIP class inhibitors

p21, p27, p57 

inhibit all CDKs, do not have specifity

binds to the Cyclin-Cdk complex, induces a conformational change by binding the active kinase, prevents substrate binding

distorts the catalytic site so it's no longer active


what are INK4 class inhibitors

p15, p16, p18, p19

specific for Cdk4, Cdk 6

binds only to the Cdk subunit, preventing it from interacting w/ the Cyclin

the inhibitor thus sequesters the Cyclin so do not have an active CDK



what does this explain


interaction btwn CDK activation and inhibition leading to activated CDK


how are cyclins degraded?

Ubiquitin pathway


what's ubiquitin?

a 76 a.a. peptide put onto proteins, used as a signal for targeting ot the proteasome 

when a protein is poly-Ubiquitinated, it becomes degraded by the proteasome 

an elaborate system causes this to occur 



what does E3 enzyme do

in the Ub-targeted degredation system, E3 is specifically responsibel for recognizing the target and putting the Ub on the target 

2 kinds of E3s: SCF and APC complexes


what is SCF complex

Ub ligase for Cyclins D, E which do G1 -> S transition 

SCF is always active but its substrates (cyclins D, E) must be phosphorylated for it to act

works during G1 phase


what's the structure of the SCF complex


what is APC complex?

anaphase promoting complex 

ubiquitin ligase that acts on Cyclin B 

is not always active; only becomes activated when Cyclin B is phosphorylated, and by the binding of a co-factor, either Cdh1 or Cdc20 

works during M phase


structure of APC complex?

Decks in MCG Class (77):