cell cycle control dec 11 2009. r.a. weinberg biology of cancer 2006. figure 8.1 the biology of...

Cell Cycle Control Dec 11 2009

R.A. Weinberg Biology of Cancer 2006. Figure 8.1 The Biology of Cancer (© Garland Science 2007)

To cycle or not

Figure 8.4 The Biology of Cancer (© Garland Science 2007)

Examples of checkpoints in the cell cycle


The restriction point

Stages during the cell cycle when pRb in under-phosphorylated

Phosphorylation of the retinoblastoma gene product is modulated during

the cell cycle and cellular differentiation

Chen PL, Scully P, Shew JY, Wang JY, Lee WH. Cell. 1989 Sep 22;58(6):1193-8.Karen Buchkovich, Linda A. Duffy and Ed Harlow Cell. 1989 Sep 22;58(6):1097-105.James A. DeCaprio, John W. Ludlow, Dennis Lynch, Yusuke Furukawa, James Griffin, Helen Piwnica-Worms, Chun-Ming Huang and David M. Livingston 1989 Cell ;58(6):1085-95.

Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 18;67(2):293-302.

Micro-injection of Rbprotein during this period blocks entryinto S phase

The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 67(2):293-302.


D and E type cyclins, which are active in early and late G1, sequentially

phosphorylate pRb (Cyclin A/B maintain pRb phosphorylation later on)


Cell cycle-dependent fluctuations in cyclin B levels


Fluctuation of cyclin levels during the cell cycle

Cell cycle-dependent phosphorylation of pRb


Control of the restriction-point transition by mitogens

Figure 8.23a The Biology of Cancer (© Garland Science 2007)

Figure 8.23d The Biology of Cancer (© Garland Science 2007)


LxCxE motif

LxCxE binding

Figure 8.24b The Biology of Cancer (© Garland Science 2007)


Control of cyclin levels during the cell cycle

CDC2 = Cdk1 is sufficient to drive the mammalian cell cycle - CDK4/6/3/2 are not required for cell proliferation and early embryogenesis - till midgestation) Santamar D, Barrie C, Cerqueira A, Hunt S, Tardy C, Newton K, Ceres JF, Dubus P, Malumbres M, Barbacid M. Nature. 2007 Aug 16;448(7155):811-5.


Actions of CDK inhibitors


Control of cell cycle progression by TGF-


Regulation of p21 localization

Figure 8.15c The Biology of Cancer (© Garland Science 2007)

Regulation of p27 localizationAKT phosphorylates p27 on T157, causing it to localize to the cytoplasm

Nat Med 2002;8:1145–52; Nat Med 2002;8:1136–44


Nuclear p27 accumulation in tumors inversely correlates with pAKT


How does p27 (and p21) regulate G1 cyclins?As opposed to their inhibitory effects on E-CDK2, they are required for D-CDK4 complex formation


Cyclin E-CDK2 facilitates its own activation by phosphorylating p27 on Thr-187, inducing its degradation

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts. Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF, Roberts JM, Sherr CJ.EMBO J. 1999 Mar 15;18(6):1571-83.



Impaired assembly of cyclin D-CDK4 complexes in MEFs lacking p21 and p27

Evidence for a role of p27/p21 as assembly factors for Cyclin D - CDK4/6 complex formation

In B - cell lysates were normalized for cyclin D1 abundanceRetrovirus encoding Flag-tagged cyclin D1



Loss of cyclin D1 and CDK4-dependent Rb kinase activity in MEFs lacking p21 and p27

GST-Rb as a substrate





Reconstitution of cyclin D1-CDK4 complexes in vivo and restabilization of cyclin

pulse-labeled for 30 min with [35S]methionine and then 'chased’ for the indicated time

QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Interestingly - Rb phosphorylation is unaffected in p21/p27 DKO MEFs - E-Cdk2 compensation

MEFs made quiescent by contact inhibition & serum starvation were trypsinized, reseeded and stimulated to enter the cell cycle in complete medium containing 10% FBS

ppRbpRb

Mouse development and cell proliferation in the absence of D-cyclins.Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, Geng Y, Yu Q, Bhattacharya S, Bronson RT, Akashi K, Sicinski P.Cell. 2004 Aug 20;118(4):477-91.

Mouse development and cell proliferation in the absence of D-cyclins.

Near normal BrdU incorporation

Heart defect in TKO

Proliferation of Cyclin D1−/−D2−/−D3−/− Cells Is Resistant to p16INK4a but Sensitive to CDK2 siRNA

Molecular Analyses of G1 Phase Progression in Cyclin D1−/−D2−/−D3−/− Cells

P27 is degraded faster in TKO, eventhough it does not formA complex with D-CDK4/6.

Perhaps it is phosphorylated more efficiently on Thr-187By Cyclin E-CDK2 - but this is not shown.

E2F regulated genes are induced but not to maximal level

pRb and p107 are phosphorylated with a delayed kinetics and not to maximal level

Positive-feedback loops and the irreversibility of cell cycle advance;E2F - cyclin E - Rb loop


cyclin E phosphorylates p27 on Thr-187 leading to its degradation



Figure 1. The Skp2 autoinduction loop. The model shows the core components of the Skp2 autoinduction loop. Arrowheads in (A) indicate the direction of flow through the loop that results in Skp2 autoinduction and promotesprogression through the restriction point. Sequential repressive effects are indicated in (B). (B) also shows the two bidirectional events within the loop. Cyclin E-cdk2 catalyzes inactivating phosphorylations of Rb while Rb Sequestration of E2F represses transcription of cyclin E. Similarly, p27 inhibits cyclin E-cdk2 activity while cyclin E-cdk2 downregulates p27 levels by catalyzing p27 phosphorylation on T187A.

The Rb - E2F - Skp2 - p27 autoinduction loop

Transc

riptio

n

Seques

trat

ion



Positive and negative regulation of the Skp2 autoinduction loop.


Cyclin D1 is targeted by multiple signaling pathways that are deregulated in human cancer

Specific protection against breast cancers by cyclin D1 ablationYu Q, Geng Y, Sicinski P.Nature. 2001 411(6841):1017-21

MMTV-Wnt1

MMTV-Neu

MMTV-Myc

MMTV-ras

Cyclin D1 is required for tumors induced by Neu and ras but not by Myc or Wnt1, which induce cyclin D2 gene expression



p53 is activated by ATM in response to DNA damage

ATM



The response to p53 induction depends on the extent of DNA damage/p53 activation and its affinity to high (pro-arrest genes) and low (pro-apoptotic) promoters

Living with p53, dying of p53Aylon Y, Oren M.Cell. 2007 Aug 24;130(4):597-600. Review.

Perturbation of the R-point transition in human tumors

In addition to cell cycle genes, Rb/E2F negatively regulates apoptotic genes

Direct coupling of the cell cycle and cell death machinery by E2FNahle Z, Polakoff J, Davuluri RV, McCurrach ME, Jacobson MD, Narita M, Zhang MQ, Lazebnik Y, Bar-Sagi D, Lowe SW.Nat Cell Biol. 2002 Nov;4(11):859-64.





Western

ChIP

Cells infected with control vector (V) or E1A-expressing (E) retroviruses

p16 is induced in aging mice

Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing.Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, Sharpless NE, Morrison SJ.Nature. 2006 Sep 28;443(7110):448-52.

p16INK4a induces an age-dependent decline in islet regenerative potentialKrishnamurthy J, Ramsey MR, Ligon KL, Torrice C, Koh A, Bonner-Weir S, Sharpless NE.Nature. 2006 Sep 28;443(7110):453-7. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a

Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT.Nature. 2006 Sep 28;443(7110):421-6.

Ageing: balancing regeneration and cancerBeausejour CM, Campisi J.Nature. 2006 Sep 28;443(7110):404-5.

http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=link&linkname=pubmed_pubmed&uid=16957737&ordinalpos=3





5-9 weeks

20-28 weeks

9-15 weeks

Improved diabetes-specific survival after STZ treatmentin p16 null mice

Nature. 2006 Sep 28;443(7110):453-7.

STZ - Streptozotocin pancreatic beta-cell specific cytotoxic drug

E2F1-/- mutant mice and transgenic mice expressingunphosphorylatable pRb are prone to cancer

Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/-)miceYamasaki L, Jacks T, Bronson R, Goillot E, Harlow E, Dyson NJ. Cell. 1996 85(4):537-48.

Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression,

precocious differentiation and adenocarcinoma

Jiang Z, Zacksenhaus E.J Cell Biol. 2002 Jan 7;156(1):185-98

The right balance is critical

Insufficient Rbcauses cancer

Too much Rbmay lead to agingas well as cancerin the long run -

- but, short termtreatment withCDK inhibitorsmay be therapeutic

Summary1. D type cyclins respond to extra cellular signals to induce Rb phosphorylation and transition through the R point.

2. Other cyclins (E, A, B) act autonomously to drive S phase and mitosis.

3. CDK inhibitors p21 and p27 are required to assemble D-CDK4/6 and inhibit cyclins E, A and B-CDK complexes.

4. As D cyclins get elevated they phosphorylate pRb (catalytic) and also sequester p21/p27 (non-Catalytic), thereby allowing E-CDK2 to further phosphorylate and inactivate pRb.

5. Cyclin A and B maintain hyper-phosphorylated ppRb through S and G2/M. At mid-mitosis pRb is dephosphorylated by type 1 phosphatase.

6. The pathways that regulate cyclins, the cyclins and associated genes are often deregulated in human cancer.

7. Rb-E2F suppress both cell cycle genes (cyclin E, Thymidylate synthase, Cdk1) as well as pro-apoptotic genes such as caspases, Puma, Noxa and Bim.

8. Loss of Rb leads to cancer, but aberrant activation of the pRb pathway may induce aging and potentially cancer in some contexts. Nonetheless, short tem treatment with CDK inhibitorsseem beneficial and various drugs are currently being investigated of the treatment of cancer.

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