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The loss of both copies of the Rbgene leads to excessivecell proliferation in the developing retina, suggestingthatthe Rb protein is particularly important for restraining ceUaivision in this tissue.The complete loss of Rb does not immediately cause increased proliferation ofAND CELLGROWTHCONTROLOF CELLDIVISIONretinal or other cell types, in part because Cdhl and CKIs also help inhibit progressionthrough Gr and in part becauseother cell tlpes contain Rb-related proteins that provide backup support in the absenceof Rb. It is also likely that otherproteins, unrelated to Rb, help to regulate the activity of E2EAdditional layers of control promote an overwhelming increase in S-Cdkactivity at the beginning of S phase.We mentioned earlier that the APC/C activator Cdhl suppressescyclin levels after mitosis.
In animal cells, however, Grand G1/S-cyclinsare resistant to Cdhl and can therefore act unopposed by theAPC/C to promote Rb protein phosphorylation and E2F-dependent geneexpression.S-cyclin, by contrast, is not resistant to Cdhl, and its level is initiallyrestrained by Cdhl-APC/C activity. However, Gr/S-Cdk also phosphorylates andinactivates Cdhl-APC/C, thereby allowing the accumulation of S-cyclin, furtherpromoting S-Cdk activation.
Gr/S-Cdk also inactivates CKI proteins that suppress S-Cdk activity. The overall effect of all these interactions is the rapid andcomplete activation of the S-Cdk complexes required for S-phaseinitiation.TheDNADamageResponseDNADamageBlocksCellDivision:Progressionthrough the cell cycle, and thus the rate of cell proliferation, is controlled not only by extracellular mitogens but also by other extracellular andintracellular mechanisms. One of the most important influences is DNA damage, which can occur as a result of spontaneous chemical reactions in DNA'errors in DNA replication, or exposure to radiation or certain chemicals.
It isessentialthat the cell repair damaged chromosomes before attempting to duplicate or segregatethem. The cell-cycle control system can readily detect DNAdamage and arrest the cycle at either of two checkpoints-one at Start in late Gt,which prevents entry into the cell cycle and into S phase, and one at the GzlMcheckpoint, which prevents entry into mitosis (seeFigure 17-21).DNA damage initiates a signaling pathway by activating one of a pair ofrelated protein kinases called ATM and ATR, which associate with the site ofdamage and phosphorylate various target proteins, including two other proteinkinases called Chkl and Chk2.
Together these various kinases phosphorylateother target proteins that lead to cell-cycle arrest.A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding a CKIprotein called p21;this protein binds to Gr/S-Cdk and S-Cdk complexes andinhibits their activities, thereby helping to block entry into the cell cycle (Figure17-63). <TGAA>DNA damage activates p53 by an indirect mechanism. In undamaged cells,p53 is highly unstable and is present at very low concentrations. This is largelybecause it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphorylation of p53after DNA damage reduces its binding to Mdm2.
This decreasesp53 degradation, which results in a marked increasein p53 concentration in the cell. In addition, the decreasedbinding to Mdm2 enhances the ability of p53 to stimulategene transcription (seeFigure 17-63).The protein kinases Chkl and Chk2 also block cell cycle progression byphosphorylating members of the Cdc25family of protein phosphatases,therebyinhibiting their function. As described earlier, these kinases are particularlyimportant in the activation of M-Cdk at the beginning of mitosis (see Figure17-25).Thus, the inhibition of cdc25 activity by DNA damage helps block entryinto mitosis (seeFigure 17-21).The DNA-damage response also detects problems that arise when a replication fork fails during DNA replication.
\{hen nucleotides are depleted, for example, replication forks stall during the elongation phase of DNA synthesis.To prevent the cell from attempting to segregatepartially replicated chromosomes, thesame mechanisms that respond to DNA damage detect the stalled replicationforks and block entry into mitosis until the problems at the replication fork areresolved.The DNA damage response is not essential for normal cell dMsion if environmental conditions are ideal. Conditions are rarely ideal, however: a low level11 0 51 106Chapter17: The Cell Cycle(discussed in chapter 20).
This loss of p53 function allows the cancer cell toaccumulate mutations more readily. Similarly, a rare genetic disease known asataxia telangiectasiais causedby a defect in ArM, one of the protein kinasesthatis activated in response to x-ray-induced DNA damage; patients with this diseaseare very sensitive to x-rays and suffer from increased rates of cancer.\Mhat happens if DNA damage is so severe that repair is not possible?Theindividual cell. Cells that divide with severeDNA damage threaten the life of theorganism, since genetic damage can often lead to cancer and other diseases.Thus, animal cells with severeDNA damage do not attempt to continue division,but instead commit suicide by undergoing apoptosis.
Thus, unless the DNAdamage is repaired, the DNA damage response can lead to either cell-cycleDNADNA damageIIATM/ATRkinaseactivation+C h k l/ C h k 2k i n a s ea c t i v a t i o nII+Mdm2PHOSPHORYLATIONOF p53stable,a c t i v ep 5 3p53 UBIQUITYLATIONAND DEGRADATIONIN PROTEASOMESA C T I V Ep 5 3 B I N D ST OR E G U L A T O RRYE G I O NO Fp 2 7 G E N Ep21 geneTRANscRrproN IVp2, mRNArnarusmloru{p21(cdki n h i b i t o rp r o t e i n )ACTIVEG1/S-Cdkand 5-LdkINACTIVEG r l S - C daknd S-Cdkc o m p l e x e dw i t h p 2 1Figure17-63 How DNAdamagearreststhe cellcyclein G1.WhenDNAisdamaged,variousproteinkrnasesarerecruitedto the siteof damageandinitiatea signalingpathwaythat causescell-cyclearrest.Thefirstkinaseat thedamagesite is eitherATMor ATR,dependingon the type of damage.Additionalproteinkinases,calledChkland Chk2,arethen recruitedandactivated,resultingin thephosphorylationof the generegulatoryproteinp53.Mdm2 normallybindstop53 and promotesits ubiquitylationanddestructionin proteasomes.Phosphorylationof p53 blocksits bindingto Mdm2;asa result,p53 accumulatestohigh levelsand stimulatestranscriptionof the genethat encodesthe CKIproteinp21.The p21 bindsand inactivatesG1lS-Cdkand S-Cdkcomplexes,arrestingt h e c e l li n G 1 .
I ns o m ec a s e sD, N Adamagealsoinduceseitherthephosphorylationof Mdm2 or a decreasein Mdm2 production,whichcausesafurtherincreasein p53 (not shown).CONTROLOF CELLDIVISIONAND CELLGROWTHarrest or cell death. As we discuss in the next chapter, DNA damage-inducedapoptosis often depends on the activation of p53. Indeed, it is this apoptosispromoting function of p53 that is apparently most important in protecting usagainst cancer.ManyHumanCellsHavea Built-lnLimitationon the NumberofTimesTheyCanDivideMany human cells divide a limited number of times before they stop andundergo a permanent cell-cyclearrest.Fibroblaststaken from normal human tissue, for example, go through only about 25-50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferationslows dolrm and finally halts, and the cells enter a nondividing state from whichthey never recover.
This phenomenon is called replicative cell senescence,although it is unlikely to be responsible for the senescence(aging) of the organism. Organism senescenceis thought to depend, in part, on progressiveoxidativedamage to long-lived macromolecules, as strategies that reduce metabolism(such as reduced food intake), and thereby reduce the production ofreactive oxygen species,can extend the lifespan of experimental animals.Replicative cell senescence in human fibroblasts seems to be caused bychanges in the structure of the telomeres, the repetitive DNA sequences andassociated proteins at the ends of chromosomes. As discussed in Chapter 5,when a cell divides, telomeric DNA sequences are not replicated in the samemanner as the rest of the genome but instead are synthesizedby the enzymetelomerase. Telomerasealso promotes the formation of protein cap structuresthat protect the chromosome ends.
Becausehuman fibroblasts, and many otherhuman somatic cells, are deficient in telomerase, their telomeres becomeshorter with every cell division, and their protective protein caps progressivelydeteriorate. Eventually,the exposed chromosome ends are sensedas DNA damage,which activates a pS3-dependent cell-cycle arrest that resemblesthe arrestcaused by other types of DNA damage (seeFigure l7-63). Rodent cells, by contrast, maintain telomerase activitywhen they proliferate in culture and thereforedo not have such a telomere-dependent mechanism for limiting proliferation.The forced expressionof telomerase in normal human fibroblasts, using geneticengineering techniques, blocks this form of senescence.Unfortunately, mostcancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergoreplicative cell senescence.ArrestorAbnormalProliferationSignalsCauseCell-CycleApoptosis,Exceptin CancerCellsMany of the components of mitogenic signaling pathways are encoded by genesthat were originally identified as cancer-promoting genes,or oncogenes,becausemutations in them contribute to the development of cancer.The mutation of asingle amino acid in the small GTPaseRas, for example, causes the protein tobecome permanently overactive,leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation.
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