LEC49: Intro to Cell Cycle

Question 1

techniques for measuring cell proliferation?

Answer 1

1) mitotic index
2) Ki-67 antigen detection
3) flow cytometry

Question 2

purpose of creating mitotic index?

Answer 2

quantitate the numebr of cells undergoing cell division

Question 3

why/how does mitotic index count work?

Answer 3

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 **

Question 4

mitotic index for proliferating tissues?

Answer 4

~10%

Question 5

calculation of mitotic index?

Answer 5

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

Question 6

what is this count

Answer 6

mitotic index

see 3 cells undergoing mitosis, cell division

Question 7

purpose of Ki-67 antigen detection?

Answer 7

detect proliferating cells

by immunohistochemistry

Question 8

where is Ki-67 expressed?

Answer 8

in cells undergoing active division

detectable using an antibody

Question 9

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

Answer 9

tissue section does not have to be fresh tissue

Question 10

what is this

Answer 10

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

Question 11

what does flow cytometry measure

Answer 11

flourescence per cell, thus measures DNA content

Question 12

how does flow cytometry to measure DNA content work?

Answer 12

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

Question 13

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

Answer 13

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

Question 14

what is flow cytometry often used to quantify?

Answer 14

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

Question 15

structure of kinases?

Answer 15

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

Question 16

what upregulates cyclin activity?

what downregulates cyclin activity?

Answer 16

upregulation of cyclin: at **transcription **level

downregulation of cyclin: at **protein degredation **level

Question 17

CDK activity?

Answer 17

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

this phosphorylation event causes movement through the cell cycle

Question 18

what are the phases of the cell cycle?

Answer 18

S, G2, M, G1

Question 19

what happens during S phase?

Answer 19

DNA synthesis:

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

Question 20

what happens during M phase?

Answer 20

mitosis

chromosomal condensation

nuclear membrane breaks down

sister chromatids separate

cytokinesis occurs

occurs through phosphorylation events

Question 21

what is the cyclin-CDK complex governing early G1?

Answer 21

Early G1: Cdk4/6, cyclin D

Question 22

what is the cyclin-CDK complex governing late G1?

Answer 22

Cdk2, Cycline E

Question 23

what is the cyclin-CDK complex governing S phase?

Answer 23

Cdk2, Cyclin A

Question 24

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

Answer 24

Cdk1, Cyclin A & Cdk1, Cyclin B

Question 25

how does a cyclin give activity to the CDK?

Answer 25

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

Question 26

what does binding of the cyclin to the Cdk do?

Answer 26

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**

Question 27

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

Answer 27

1) cyclin levels 2) phosphorylation of the CDK itself - activating & inhibiting phosphorylation 3) inhibitors

Question 28

is cyclin binding necessary/sufficient for Cdk activation?

Answer 28

cycling binding is **necessary but not sufficient for Cdk activation**

Question 29

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

Answer 29

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**

Question 30

describe the process of activating phosphorylation

Answer 30

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

Question 31

what confers inhibitory phosphates

Answer 31

Wee1 enzyme

Question 32

describe process of inhbitory phosphorylation and how it is counteracted

Answer 32

**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**

Question 33

what counteracts inhibitory phosphorylation activity on Cyclin-CDK complexes?

Answer 33

Cdc25 family phosphatases: Cdc25A, B C

Question 34

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

Answer 34

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**

Question 35

classes of protein inhibitors of CDKs?

Answer 35

1) CIP family 2) INK4 family

Question 36

what are CIP class inhibitors

Answer 36

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**

Question 37

what are INK4 class inhibitors

Answer 37

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**

Question 38

what does this explain

Answer 38

interaction btwn CDK activation and inhibition leading to activated CDK

Question 39

how are cyclins degraded?

Answer 39

Ubiquitin pathway

Question 40

what's ubiquitin?

Answer 40

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

Question 41

what does E3 enzyme do

Answer 41

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

Question 42

what is SCF complex

Answer 42

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

Question 43

what's the structure of the SCF complex

Answer 43

Question 44

what is APC complex?

Answer 44

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

Question 45

structure of APC complex?

Answer 45