LEC49: Intro to Cell Cycle Flashcards Preview

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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



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