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
see 3 cells undergoing mitosis, cell division
purpose of Ki-67 antigen detection?
detect proliferating cells
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
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 polymerase, ntds are needed for DNA polymerase to make more DNA
what happens during M phase?
nuclear membrane breaks down
sister chromatids separate
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
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
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?
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?