Kleinsmith_ch2

# Kleinsmith_ch2 - FEATURES OF CANCER CELL PROLIFERATION The...

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Unformatted text preview: FEATURES OF CANCER CELL PROLIFERATION The concept of uncontrolled proliferation is unique to multicellular organisms. In most single—celled organisms. such as bacteria or yeast. the presence of sufficient nutri— ents in the surrounding environment is the main factor that determines whether cells will grow and divide. the situation is reversed in multicellular organisms; cells are usually surrounded by nutrient—rich extracellular fluids, but the organism as a whole would be quickly destroyed if each cell were to continually grow and divide just because it had access to adequate nutrients. (lancer is a potentially lethal reminder of what happens when cell proliferation continues unabated without being coordinated with the needs of the organism as a whole. Cancer Cells Produce Tumors When Injected into Laboratory Animals it is the loss of normal growth control that causes cancer cells to produce a continually growing mass of tissue—in other words, a tumor——but uncontrolled growth does not mean that tumor cells always divide more rapidly than normal cells. 'I‘umors can grow slowly, quickly, or some— where in between. The distinctive feature of tumor growth is not the speed of cell division but its uncontrolled nature. in contrast to the proliferation of normal cells, where cell division and cell dit'lerentiation are kept in proper balance. this finely [Lined arrangement is disrupted in tumors and cell division is uncoupled from cell differentiation. thereby leading to a progressive increase in the number of dividing cells (see Figure |~4. bottom]. To determine experimentally whether a particular cell behaves in this way. the cell must be injected into an appropriate host organism to see if a tumor will develop. l-preriinents involving animal cells are fairly straightfor— ward because the cells can simply be injected into animals ol'the same genetic type. The situation with htllnan cells is more complicated. Injecting human cancer cells into other humans for testing purposes would be unethical. and using standard laboratory animals is not reliable: An animal‘s immune system is likely to reject human cells because they are of foreign origin. One way around this obstacle is to inject human cells into mutant strains of mice whose immune systems are unable to attack and destroy foreign cells. When human cancer cells are injected into such immunologically deficient animals. the cells will usually grow into tumors without being rejected. Cancer Cells Exhibit Decreased Density-Dependent Inhibition of Growth .-\lthough studying cancer cells in intact organisms is useful for investigating solne of the properties of malignant tumors. issues related to the control of cell proliferation are often easier to investigate in cells grown under artificial laboratory conditions. In such cell culture studies. cancer 18 Chapter 2 Protite ot a Cancer Cell cells are isolated from a tumor and placed in a defined growth medium containing nutrients. salts. and other molecules required for cell growth. The main reason for studying cells in culture is that this method allows the cells to be observed under carefully controlled conditions where their behavior can be assessed without the complicating effects of secondary factors present in an intact organism. When most types of normal cells are placed in a culture vessel [test tube. bottle, flask. or dish} and then covered with an appropriate growth medium, they divide until the surface of the container is covered by a single layer of cells. When this monot’rn-‘er stage is reached. cell movements and cell division both tend to stop. in the early 1950s. Michael Abercrombie and loan l'ieaysman intro- duced the term “contact inhibition " to refer to the decrease in cell motility that occurs when cells make contact with one another in culture. The salne term has also been used to refer to the inhibition ol‘ cell division that takes place when culture conditions becolne crowded. Because of the confusion that can result from the double meaning or this term, the phrase density-dependent inhibition of growth is now routinely used when referring to the inhibition of cell division that occurs in crowded cultures. In contrast to normal cells, cancer cells do not stop dividing when they reach the monolayer stage. instead, they continue to divide and gradually pile up on top of one another, forming multilayered aggregates [Figure 2—1]. in other words. cancer cells are less susceptible to density— dependent inhibition of growth than are their normal counterparts. The relationship between the tendency of cancer cells to grow to high population densities in culture and their ability to form tumors has been investigated using cancer cells that differ in their susceptibility to density- dependent inhibition of growth. (“.ells that are very sensitive to density—dependent inhibition can be produced by growing cells under uncrowded conditions; every time the population density increases and crowding is imminent. the cells are simply diluted and transferred to a new culture flask. Cells obtained in this way will not grow to high pova lation densities in culture. Alternatively. cell populations that are insensitive to density-dependent inhibition of growth can be produced by consistently growing cells in overcrowded conditions. Such cell populations become less susceptible to density—dependent inhibition of growth. reaching much higher population densities before cell divi— sion stops. ‘t\"l1en these different cell populations are tested for their ability to produce tumors in mice. ttunor—forn'ting ability is found to be directly related to the loss of density- dependent growth control; in other words. cells capable of growing to the highest population densities in culture are most effective at forming tumors in animals {Figure 2—3}. Cancer Cell Proliferation Is Anchorage-Independent Another way in which the proliferation of cancer cells differs from that of normal cells involves the requirement for anchorage. Most normal cells will not proliferate if i ﬁrm; (J5 i Reheat cells - 47/ K’s?» ‘33; ‘1 Place cells in flask 1c.— 3/? i If, If \._\\ If!» Ci}. (3: CE} "-I I @ i'. (a wtge/ at ﬁﬁqv ~-Q2_.. - 2c.._ 3““ Cells attach to / bottom of flask and start dividing r " Cells continue dividing, ft Cells stop dividing f piling up on one another f i ' at monolayer stage Figure 2—1 Density-Dependent inhibition of Growth. When normal cells are placed in culture. they tend to divide until the surface of the container is covered by a single layer of cells tthe monolayer stage i. Theinhibition of further cell division that occurs at the crowded. monolayer stage is called dertsity—dependent inhibition ofgrowth. In contrast to the behavior of normal cells, the proliteration ofcanccr cells tends to continue beyond the ntonolayer stage as dividing cells pile tip on top of one another. forming nulltilayered aggregates. they are put in a liquid growth medium and shaken or stirred to keep them in suspension, nor will they prolif- erate if they are placed in a semisolid medium such as soft agar. When they are provided with an appropriate solid surface to which they can adhere. however, the cells will attach to the surface, spread out, and begin to proliferate (Figure 2-3). The gi'o\-vth of normal cells is therefore said to be anchorage-dependent. in contrast, most cancer cells grow well not just when they are anchored to a solid surface, but also when they are suspended in a liquid or semisolid medium. The growth of cancer cells is therefore said to be anchorage-independent. In intact organisms, the requirement that cells be anchored before they can reproduce is met by binding cells to the extracellular matrix, an insoluble meshwork of protein and polysaccharide tibers that fills the spaces between neighboring cells. Cells attach themselves to the extracellular matrix through cell surface proteins called inregriris, which bind to molecules present in the matrix. If this attachment is prevented experimentally using chemicals that block the binding of integrins to the matrix, normal cells are prevented from dividing and may even commit suicide by opoptosis, an orchestrated program for cell death described later in the chapter. r‘ipoptotic cell death triggered by lack of contact with the extracellular matrix is called anoikis l{from the (ireek word for “ltoittelessttess"l. Anoikis is an important safeguard for maintaining tissue integrity because it prevents normal cells from floating away and setting up housekeeping in another tissue. The lack of anchorage simply causes cells to commit suicide along the way, (lancer cells are not subject to this normal safeguard because they are anchorage—independent and so can spread to distant sites without self—destructing. Considerable evidence suggests that anchorage— independent growth exhibited by cells grown in culture is related to their ability to form tumors. One set of studies involved cells with many of the traits of cancer cells, including decreased density—dependent inhibition of growth, low requirements for external growth factors (p. 22), and anchorage—independent growth. Single cells were isolated from the original population and allowed to proliferate separately, thereby creating a series of clones, which are individual cell populations each derived from the proliferation of a single cell. Careful analysis of the clones revealed that some of them had lost one or more of the initial properties. When the ability of these clones to produce tumors in animals was compared, anchorage-independent growth was the only _. .- N O U1 D O C) 0 U1 0 Days required for 50% of injected animals to develop tumors . i r 5 '10 15 Cell density at which growth stops in culture (cellsicm2 x105} Figure 2—2 Tumor-Forming Ability of Mouse Cells That Differ in Their Susceptibility to Density-Dependent Inhibition of Growth. liach point represents a different cell population. The data show that cell lines that grow to higher population densities in culture—that is, cells that are less susceptible to density-depentlenl inhibition ofi‘srtnvth—tcnd to produce tuntors more rapidh when iniected into mice, Note that the _'l'- axis is measuring how long it takes for tumors to develop. so higher \alues indicate cells that are less efficient at forming tumors. :.\Ll.l]‘lL‘tl Iiom .s'. .\. -\.It'ttll~n\it and (i. I. 'l'otiaro, .h't'ii'in'x' in." I Not-i I: lli.‘.-l_! Features oi Cancer Cell l-‘Jrolileration 19 Place cells in flask f:- r r K. ‘H ‘5 __<I___ _ / Allow cells to settle xiii—Z 5.1? Keep cells suspended by shaking or using soft agar Cells grow WELL Cells grow POORLY Figure 2-3 Anchorage-Dependent and independent Growth. _ Cancer cells Place cells in flask Keep cells suspended by shaking or using soft agar Allow cell to settle P’s‘l’: %A\. “be c. in. when 3" «£3 5’" _,.. Cells grow WELL Cells grow WELL When normal cells are placed in culture, they only grow well when they are allowed to adhere to the surface ot‘the culture llask; their growth is therefore said to be anchorage—det‘lendent. In contrast, cancer cells grow well not just when they are anchored to a solid surface, but also when they are freely suspended in a liquid or a semisolid medium. The growth ol‘cancer cells is therefore said to be anchorage—independent. property consistently retained by all the clones that could produce tumors. In other words1 the ability to form tumors appeared to require cells whose growth in culture is anchorage-independent. This connection between anchorage—independent growth and tumor formation is not without its exceptions, however. Some cells exposed to cancer—causing chemicals have been found to exhibit anchorage—independent growth in culture but do not form tumors when injected into animals. in addition, studies involving a long—term culture of mouse cells, which were anchorage—dependent and unable to form tumors in animals, showed that the cells could acquire the capacity to form tumors if they were attached to glass beads prior to being implanted in mice. Such observations indicate that despite its general association with the ability to form tumors, anchorage- independent growth in culture is not an absolute prerequisite for tumor formation. Mechanisms for Replenishing Telomeres Make Cancer Cells Immortal One of the most striking differences between normal cells and cancer cells involves their reproductive lifespans. When normal cells are grown in culture, they usuthr divide for only a limited number of times. For example, human ﬁbroblasts—a cell type whose behavior has been extensiver studied—divide about 50 to 60 times when placed in culture and then stop dividing. undergo a variety 20 Chapter 2 Profile of a Cancer Cell oi degenerative changes, and may even die {Figure 2-4). Cancer cells exhibit no such limit and continue dividing indeﬁnitely, behaving as if they were immortal. A striking example is provided by Heir: cells, which were obtained from a malignant tumor of the uterus arising in a woman Figure 2-4 Microscopic Appearance of Young and Old Human Fibroblasts Growing in Culture. (Left) Young ﬁbroblasts that have divided a relatively small number of times in culture exhibit a thin, elongated shape. {Right} After dividing about St} times in culturei the cells stop dividing and undergo a variety ofdegenerative changes. Note the striking difference in appearance between the young (dividing) and older {nondividing} cells. [Courtesy ol- |.. I |a_vflick.| named Henrietta Lacks (hence the name “HeLa” cells). After removing the tumor in a cancer operation performed in 1951, doctors placed some of its cells in culture. The cultured cells began to grow and divide and have continued to do so for more than 50 years, dividing more than 18,000 times with no signs of stopping. Why are cancer cells capable of reproducing indeﬁ- nitely in culture, whereas most normal human cells divide no more than :30 or 60 times? The answer is related to the mechanism by which cells replicate their DNA. Each time a cell divides, its chromosomal DNA molecules must be duplicated so that a complete set of genetic instructions can be distributed to each of the two cells produced by cell division. However, the biochemical mechanism responsible for DNA replication has an inherent limitation: The enzymes that replicate DNA are unable to copy the very end of a linear DNA molecule, perhaps the ﬁnal 50 to 100 nucleotides or so. As a result. each time a DNA molecule is replicated. it is in danger of losing a small amount of DNA at each of its two ends. If this trend were to continue indeﬁnitely, DNA molecules would become shorter and shorter until there was nothing left, and we would not be here today! To solve this so—called end-replication problem. cells place a special type of DNA sequence at the two ends, or telomeres. of each chromosomal DNA molecule. The special DNA consists of multiple copies of a short base sequence repeated over and over again. For example. in humans the six-base sequence TTAGGG is repeated about —TTAGGG TTAGGG TTAGGG— —AATCCC AATCCC AATCCC- 1L: Telomere sequences DNA Repeated cell divisions =. Repeated cell divisions ﬂ 1 l l l l l 1 Figure 2-5 Telemere Structure in Human DNA. the same sequence repeated over and over, the end of one 2500 times in a row at the ends of each chromosomal DNA molecule at birth. These telomere sequences are pro- tected by telomere capping proteins. and the DNA also loops back upon itself to protect the end of the chromo- some even further (Figure 2—5]. Unlike genes, whose DNA base sequences code for useful products. telomeric DNA does not code for anything but simply consists of the same six-base sequence repeated again and again. Placing such noncoding telomere DNA at the ends of each chromo— some ensures that a cell will not lose any important genetic information when DNA molecules are shortened slightly during replication. Since telomcres get shorter with each cell division, they provide a counting device for tracking how many times a cell has divided. If a cell divides too many times, the telomeres become extremely short and are in danger of disappearing entirely. When this happens. the telo— meric DNA becomes too short to bind telomeric capping proteins or generate a loop, exposing a bare end of double—stranded DNA. Such unprotected DNA ends are very unstable and often fuse with each other, creating joined chromosomes that tend to become fragmented and separate improperly at the time of cell division. In normal cells, such a hazardous outcome is prevented by a mechanism in which the unprotected DNA at the end of a chromosome triggers a pathway that halts cell division or triggers cell death. This pathway helps protect organisms from any inappropriate, excessive proliferation of adult cells. Telomere capping proteins A... m v The six—base lelomere sequence. 'l'TAtitiG. is repeated about 2500 times in a row at the ends of each chromosomal DNA molecule at birth. Because telomeric DNA contains DNA strand can loop back and form base pairs with an earlier repeal ofthe same complementary sequence in the opposite strand. 'l'elomeres get shorter with each cell division, thereby providing a counting device for tracking how many titnes a cell has divided. If a cell divides too many times. the telomeres become too short to generate a loop or bind telomeric capping proteins. Such unprotected DNA ends are unstable and trigger pathways that lialt cell division or trigger cell death. 21 Features of Cancer Cell Proliferation h- " . | “ But what happens with cells that must divide for pro— longed periods of time, such as the germ cells that give rise to sperm and eggs or the bone marrow cells that continu— ally produce new blood cells? Such cells prevent excessive telomere shortening, by producing an enzyme called telomerase, which adds new copies of the telomeric repeat sequence to the ends of existing DNA molecules. The telomerase—catalyzed addition of new telomere repeat sequences prevents the gradual decline in telomere length that would otherwise occur at both ends of a chromosome during DNA replication. The presence of telomerase therefore allows cells to divide indeﬁnitely without telomere shortening. How do the preceding considerations apply to cancer cells? If cancer cells behaved like most normal cells, which do not produce telomerase, repeated cell divisions would cause the telomeres to become unusu- ally short and the cells would eventually be destroyed. Most cancer cells circumvent this problem by activating the gene that produces telomerase, thereby causing new copies of the telomeric repeat sequence to be continually added to the ends of their DNA molecules. A few cancer cells activate an alternative mechanism for maintaining telomere sequences that involves the exchange of sequence information between chromo— somes. By one mechanism or the other, cancer cells maintain telomere length above a critical threshold and can therefore divide indefinitely. GROWTH FACTORS AND THE CELL CYCLE We have now seen that cancer cells differ from most normal cells in that they grow to high population densities in culture, exhibit anchorage-independent proliferation, and divide indefinitely because they possess mechanisms for maintaining telomere length. These traits play an important permissive role in allowing cancer cells to continue dividing, but they do not actually cause cells to divide. The driving force for ongoing proliferation can be traced to abnormalities in the signaling systems that control cell division, the topic to which we now turn. Cancer Cells Exhibit a Decreased Dependence on External Growth Factors The cells of multicellular animals do not normally divide unless they are stimulated to do so by an appropriate signaling protein known as a growth factor. For example, if cells are isolated from an organism and placed in a culture medium containing nutrients and vitamins, they will not proliferate unless an appropriate growth factor is also provided. Growth media are therefore commonly supplemented with blood serum, which contains several growth factors that stimulate cell proliferation. One is platelet-derived growth factor (PDGF), a protein pro- duced by blood platelets that stimulates the proliferation 22 Chapter 2 Proﬁle of a Cancer Cell of connective tissue cells and smooth muscle cells. Another growth factor in blood serum, called epidermal growth factor (EGF), is also widely distributed in tissues. Some growth factors, such as EGF, stimulate the growth of a wide variety of cell types, whereas others act more selec- tively on particular target cells. Growth factors play important roles in stimulating tissue growth during embryonic and early childhood development, and during wound repair and cell replacement in adults. For example, release of the growth factor PDGF from blood platelets at wound sites is instrumental in stimulating the growth of tissue required for wound healing. Growth factors exert their effects by binding to receptor proteins located in the plasma membrane that forms the outer boundary of all cells. Different cell types have different plasma membrane receptors and hence differ in the growth factors to which they respond (Figure 2-6). The binding ofa growth factor to its corre- sponding receptor triggers a multistep cascade in which a series of signal transduction proteins relay the signal throughout the cell, triggering molecular changes that stimulate (or occasionally inhibit) cell growth and divi- sion. Cells will not normally divide unless they are stimulated by an appropriate growth factor, but this restraint is circumvented in cancer cells by various mech- anisms that create a constant signal to divide, even in the absence of growth factors. Some cancer cells achieve this autonomy by pro- ducing their own growth factors, thereby causing cell proliferation to be stimulated without the need for growth factors produced by other cells. Similarly, other cancer cells possess abnormal receptors that are perma- nently activated, causing cell division to occur whether growth factors are present or not. Cancer cells may also produce excessive quantities or hyperactive versions of other proteins involved in relaying signals from cell surface receptors to the cell division machinery in the cell’s interior. The net effect of the preceding types of alterations is to cause the pathways that signal cell proliferation to become hyperactive or even autonomous, functioning in the absence of growth factors. In Chapters 9 and 10, the molecules involved in these pathways and the ways in which they malfunction in cancer cells will be described in detail. The Cell Cycle Is Composed of G1, 5, G2, and M Phases To understand how pathways activated by growth factors ultimately cause a cell to divide, it is first necessary to review the events associated with cell division. in cells that are dividing, the nuclear DNA molecules must be duplicated and then distributed in a way that ensures that the two new cells each receive a complete set of genetic instructions. In preparing for and accomplishing these tasks, cells pass through a series of discrete stages called G1 phase, S phase, G2 phase, and M phase. l Normal cells Signal / Nucleus ® W transduction___ - proteins " @ E31 4 CELL DIVISlON EGF receptor c PDGF Plasma membrane receptor .\ / (ELL DIVlSION _._._p -- - _ _ Cancercell Mutant receptor \ CELL DIVISION Figure 2'5 Growth Factor Signaling. Ul'ganistris produce do;ch oldil't'erent growth factors. each stimulating the prolileration oi target cells that contain .1 receptor to which the growth tat tor cart bind. the top two cells contain receptors tor epidermal growth factor [litit- I and platelet derived grow 1h lactor : PI )1 il' :. respectively. The hurtling 111 a grow 1h lactor to its correspondith receptor triggers changes in signal ll'.tllsLll|c'lltlll proteins that cause cells containing that particular receptor to di\ ide. 'l'lte cancer tell shown .11 the lioltom contains a mutant receptor that is locked in an active conligttl'atlon. causing the cell to cotitintrally divide :even in the ahsence ot'grow 1h tactor I. .-\|1er.11ions in other proteins ll'l\"ttl\L‘Ll in growth tactor signaling pathway» can likewise cause cancer cells to tll\ ide in .111 uncontrolled I'asltion. The signal transduction proteins involved in such pathways will lie descrihed in Chapters 1! and it] .ey.‘ see i igure tJ- ts' I. The tour phases are collectively reterred to as the cell cycle tl‘igure 2 7- _: (it—the lirst phase to occur alter a cell has iust divided lasts ahout H to 11] hours in human cells. hut rapidly varies the most in length. .-\ typical til phase dividing cells Iiiay spend only a few minutes or hours in (ll. Conversely, cells that divide very slowly may become arrested in (11 and spend weeks, months. or even years in the offshoot of (it called the GO phase it} zerol. After completing (it, the cell enters S phase. a period 111' roughly (1 to 8 hours' when the chromosomal DNA molecules are replicated. Next comes (i2 phase. where .i to -1 hours are spent making linal preparations l‘or cell division. The cell then enters .\I phase, which takes ahout ati hour to physically divide the original cell into two new cells. 'l'he main events ol' M phase include division ot' the nucleus or mitosis. l‘ollowed by division ol‘ the cytoplasm‘ or cytokincsis. The two newly formed cells then enter again into til phase and hegin preparations tor another round (it cell division. laken together. the (ii. Sc and (i2 phases are collec— tively relerred to as interphase. Besides providing the time needed lor a cell to make copies of its l).\'.-\ molecules. interphase is also a period (it cell growth. lnterphase occu- pies ahottt 93% of a typical cell cyclcx whereas the actual process ol‘ cell division [M phase] only takes about 3"”. Uvet'alL the time occupied hy the various stages ol‘ the cycle allows a typical human cell to divide as ol'ten as once every Is to It hours. However. the various cell types that make up the hody dill'er greatly in cycle time. ranging from cells that divide very rapidly and continttottsly to dit'l'erentiated cells” that do not divide at all. the yariahility ohserved in rates of cell division means that mechanisms must exist lor regulating progression through the cell cycle. A key control point ltas heen identitied during late til, where the cell cycle is usually halted in cells that stop dividing. lior esaniple. the division ol' cultured cells can he slowed down or stopped by allowing the cells to ru 11 out 111' either nutri— ents or growth lactors, or hv adding inhiliitors ot vital processes such as protein synthesis. [11 such cases‘ the cell cycle is halted in late til at a point rel—erred to as the restriction point, l'nder normal conditions, the ahility to pass through the restriction point is governed mainly by the presence til growth l‘actot's. (.iells that sticcesslttlly move through the restriction point are committed to S phase and the ret‘nainder ol' the cell cycle. whereas those that do not pass the restriction point enter into tit} and reside there l'or variahle periods ol' time. awaiting a signal that will allow them to re-enter ti] and pass through the restriction point. Progression Through the Cell Cycle ls Driven by Cyclin-Dependent Kinases .-\t the molecular level. passage through the restriction point and other key points iii the cell cycle is controlled in proteins known as cyclin—dependent kinases {(Idks]. (jdks are protein kinases. a term reterring to a class or en'xymes that regulate the acti\ ity oi" targeted protein mol— ecules hy catalyxing-their phosphorylation I'altacliment ol— phosphate groups to the targeted proteinst. During protein phosphorylation reactions. the phosphate group is 'C'ir-:::'-:-.-'tl1 i'.:1;:1;.‘.-r. :1: .' ("rel (ls-Him 23 DNA per cell DNA synthesis / Cal 5 62 M Gt S 62 | I T‘__"—l— 10 2t) 30 40 Hours Mitosis [nuclear division} and Cytokinesis (cytoplasmic division] Restriction point Figure 2-7 The Cell Cycle. The graph on the left shows how the four phases ol—the cell cycle [ii I . 5. (i3. and M] are deﬁned by two variables: DNA synthesis and the amount of DNA per cell. 5 phase is delined as the time during the cell cycle when DNA synthesis is taking place. leading to a doubling of the amount ol‘ DNA per cell. M phase is the time when the amount of DNA per cell drops in halfas cells divide. (it is delined as the interval between M phase and S phase. and (i2 is delined as the interval between S phase and M phase. The cell cycle is commonly represented with a circular diagram like the one shown on the right. The restriction point is a control point near the end (Ii-(ii where the cell cycle can he halted until conditions are suitable for progression into S phase. Under normal conditions. the ability to pass through the restriction point is governed mainly by the presence oligrotvth factors. donated to the targeted protein by the high—energy cont— pound ATP (adenosine triphosphate}, which is converted to ADP (adenosine diphosphate] during the reaction. Cells contain dozens of different protein kinases, each designed to regulate the activity of a speciﬁc group of proteins by catalyzing their phosphorylation. As the name implies, a cyclin-dependent kinase (Cdkl only exhibits protein kinase activity when it is bound to another type of protein called a cyclin. Progression through the cell cycle is controlled by several (Idks that bind to different cyclins, thereby creating a variety of Cdk- cyclin complexes. (.Zyclins involved in regulating the progression from (it to S phase are called Gt cyciiits, attd the Cdk molecules to which they bind are known as GI (Stilts. Likewise, cyclins involved in regulating passage from (“:2 into M phase are called mitotic cyciirts, and the Cdk molecules to which they bind are known as ittitotic Criks. Cdk—cyclin complexes act by phosphorylating specific target proteins whose actions are required for various stages of the cell cycle. How do (Idk—cyclin complexes ensure that passage through key points in the cell cycle only occttrs at the appropriate time? In addressing this question, we will briefly consider the behavior of the mitotic Critic-cyclin roiiiplex (mitotic (Idk bound to mitotic cyclin), which regulates passage from G2 to M phase. Mitotic cyclin is continuously synthesized throughout interphase and grad- ually increases in concentration during (:1, S, and G2, eventually reaching a concentration that is high enough to bind to mitotic (ldk (Figure 2-8). The resulting mitotic (Ldk-cyclin triggers passage from 62 into M phase by phos- 24 Chapter 2 Profile of a Cancer Cell phorylating key proteins involved in the early stages of mitosis. For example, proteins phosphorylated by mitotic (Idk—cyclin trigger nuclear envelope breakdown, chromo- some condensation, and mitotic spindle formation. Shortly thereafter, mitotic cyclin is targeted for degrada— tion by an enzyme called the rirmpltrtsc-promotiiig complex and mitotic (Jdk becomes inactive, triggering the exit from mitosis. During the next cell cycle, mitosis cannot be triggered until the concentration of mitotic cyclin builds up again. Besides being regulated by the availability of cyclins, the activity of the various Cdkvcyclin complexes is controlled by reactions in which Cdk molecules are altered by pituspimrylrititm (addition of phosphate groups} attd depltosphoryiution (removal of phosphate groups). Figure 2-9 illustrates how the mitotic Cdk—cyclin complex is regulated in this way. In step (13-, the binding of mitotic cyclin to mitotic Cdk creates a complex that is initially inactive. Before it can trigger passage from G2 into M phase, the complex requires the addition of an activating phosphate group to a particular amino acid of the Cdk molecule. Prior to adding this phosphate, however, inhibitory phosphate groups are ﬁrst attached to the Cdk molecule at two other locations, preventing the Cdk from functioning (step {23}. The activating phosphate group, highlighted with yellow in step (33-, is then added. The last step in the activation sequence is the removal of the inhibiting phosphates by a speciﬁc enzyme called a protein phosphatase (step '14)). Once the phosphatase begins removing the inhibiting phosphates, a positive feedback loop is set up: The activated Cdk—cyclin complex f I '! Anaplrzrw promoting K complex Ii" } ldeqradas cyclinr If Degraded . ._. r_ clin rc y Cdk U (32 M Transition Q " I .\d$\\ C . i l'” fun: entra'uol‘ D" Figure 2-8 The Mitotic Cdk Cycle. 'l'liix diagram illtixlrdix'x llik' usiilrul til initcslit (ill; in mitotic zi'clinduring[Eu-wlqulta l}1||'l|1:_[(il..\-\i1|1(l til. llrt- [iilll1[l\ \ it lin t'iint't-nlrulii-n gradually ink-l-L'tihL". \mr' [lii- i-nil iil' t i_‘. mitotic ( :tiL .mtl ru'lin form .in iiriiw mml‘lcx llml .. triggers [irismigc [i'mti t if. llllt‘ .\l plmw |\._\' [‘l'mh]\l1iil'_\l.l[li1g print-i215 inmli L‘tl in tlu‘ mitotic : events llhlk'Ll. [in tirnlr'ilruling; tn .1L'li\.llltl]1 ul' [lic .rnriphtm‘ promining armpit-x. \rlrirlr dcgrmlr'hr‘ytlili. ‘ilic mitotic {.le L'rL'liu. mmplcx tilxn liringa .ilmut ita im'rr LlL‘IlllHL‘. allim'ing .f thct‘nmplminn Ul- mum-ix .intl untrr into t ii iii the m‘xl t'cll Li'tig'. " -| l ? '.-hTP.;'- —-.ims;r - _ --—_---_-------: . 2 ADP ADP _ . .. .. : i ' 2 P i Inhibiting kiimsm Antiviitirrg Swims“ Plioﬁphatasc- Mitotic Cdk- Plil'lspiliorylatﬁfl ° . Mitotit Cdk cyclin complex mmpit-x cyclin complex I :‘iNAC' ithi iINACTIVEJ [ACTIVE] E) ' ' Figure 2-9 Regulation of Mitotic Cdk-Cyclin by Phospharylation and Dephusphorylatinn. .\L[i\¢tii1‘li .i:' lllilllllx' (Idk-L‘rrlin inmlrca llu- .rililiiiirn til ll2l1ll1liiligdnll .iclimting [‘litthl‘liillL‘ grinnm l'ullmwtl l\_\ rcmuml irl' lhu iirliiluiin}; phosphate group». li_\' .i l‘lit‘kpliillilht'. t illu‘ rt-miirnl irl' llrt- inhibiting plrmplmlr groups htix lug-ran in nu? -'- \ tr pumm- fi't’tllhik‘ii limp ix wl up: 'l'ht- .iL'iiuiit-rl t :tll\' cuiin yum]?ch gL‘llL‘l'tllL‘Ll l)‘.' Il‘in TL‘tlL'llUll niniultrim [lig plriixplrmm ilri-rx-l“. causing 1l1L' ilL'lirnEiLJn l‘riu'uax 1:: prriL'L‘t'il rmrri.‘ lupitllL I" ,=-'-; gl!‘;-Zl ‘.| ii'= i' I. :- -2 'Ja-i". L": 25 generated by this reaction stimulates the phosphatase, thereby causing the activation process to proceed more rapidly. After being activated, the mitotic. (Idk—cyclin complex triggers passage from ($2 into M phase by catalyzing the phosphorylation of proteins required for the onset of mitosis. Growth Factor Signaling Pathways Act on the Restriction Point by Stimulating Phosphorylation of the Rb Protein Now that (Idk—cyclins have been introduced, we can explain how growth factors exert their control over cell proliferation. lf normal cells are placed in a culture medium containing nutrients and vitamins but no growth factors, the cells become arrested at the restriction point. Subsequent addition of growth factors is sufficient to cause the cells to start dividing, again. How do growth factors cause (ii—arrested cells to resutne progression through the cell cycle? The binding of a growth factor to its corresponding cell surface receptor causes the receptor to become activated, and the activated receptor then triggers a complex pathway of reactions involving dozens of different cytoplasmic and nuclear molecules that relay the signal throughout the cell. A detailed description of the molecules involved in growth factor signaling pathtx-ays will be provided in Chapters 9 and It), when we discuss how cancer—causing mutations affect components of these pathways. For now we are con— cerned only with the question: How do these pathways Plasma membra ha y / -/' CELL INTERIOR / impinge on the cell cycle and cause cells to pass through the restriction point and into S phase? The answer to this question is that growth factor signaling pathways trigger the production of (Idkw‘yclins that in turn catalyze the phosphorylation of target pro- teins required for the transition into S phase. A key target is the Rb protein, a molecule that normally restrains cell proliferation by preventing passage through the restric- tion point (figure 2—10}. After (Idk—cyclin phosphorylates Rb, it can no longer exert this inhibitory influence and cells are free to pass through the restriction point and into S phase. The mechanism by which the Rb protein exerts its inhibitory control over the restriction point will be described in Chapter 10, where we examine the role played by Rb mutations in the development of cancer. Checkpoint Pathways Monitor for DNA Replication, Chromosome-to-Spindle Attachments, and DNA Damage The ability of growth factors to promote passage through the restriction point by stimttlating the production of (de-cyclins that phosphorylate Rb is just one example of how the cell cycle is controlled by external and internal factors that determine whether or not a cell shouch divide. Another type of cell cycle control involves a series of checkpoint pathways that prevent cells froin proceeding from one phase to the next before the preceding phase has been properly completed. These checkpoint pathways monitor conditions within the cell and transiently halt the l Restriction point l St nal ‘ transduction Stop. ' ' 61—» s Q l proteins )fcoa _’ _b _* _. sir «I: l .J K Cdk—qdm Proceed: \ Gl——DS Growth \- factor \\ \ ADP __.-\\H 0 Raiding oi ty'ou-vtli factor to receptor leads to production of active Cdk (.yclin. donor. l 9 Cdk cyclin adds phosphate groups to Rb protein. using A lP as phosphate group | 6 After being phospltol'ylated. the Rb protein tan no longer exert its Inhibitory influence on the restriction point and cells are free to pass into S phase. Figure 2-10 The Rb Protein and Restriction Point Control. The Rh protein imnmtly prevents cells from passing through the restriction point in the absence an appropriate growth factor. Growth factors trigger the production ofactive (Idk—cyclin complexes that phosphorylate the Rb protein. After being phosphorylaled, the Rb protein can no longer exert its inhibitory influence on the restriction point and cells are free to pass into S phase. {As will be described in Chapter It}, the Rb protein exerts its effects by binding to a protein called lilli, which regulates gene expression lsee Figure Ill—3].] 26 Chapter 2 Profile of a Cancer Cell cell cycle at various points if conditions are not suitable for continuing {Figure 2-1 1}. One such mechanism, called the DNA replication checkpoint, monitors the state of DNA replication to ensure that DNA synthesis has been completed prior to proceeding with cell division. If DNA replication is not complete, the cell cycle is halted to allow DNA replication to be ﬁnished prior to entering M phase. The existence of the DNA replication checkpoint has been demonstrated by treating cells with inhibitors of DNA synthesis. Under such conditions the ﬁnal dephosphorylation step involved in activating mitotic Cdk~cyclin (step is") in Figure 2-9} is blocked through a series of evettts triggered by proteins associated with replicating DNA. The resulting lack of active mitotic Cdk-cyclin halts the cell cycle at the end of G2 until DNA replication is completed. A second checkpoint mechanism. called the spindle checkpoint, acts between the memphnse and mmphose stages of mitosis, the point where the two duplicate sets of chromosomes are about to be parceled out to the two new cells being formed by the process of cell division. At the end of metaphase, the two sets of chromosomes are nor— mally lined up at the center of the mitotic spindle, a structure composed of nticrotubules that attach to the chromosomes and eventually pull them into the two newly forming cells. Before chromosome movement begins (the event that marks the beginning of anaphase), the spindle checkpoint mechanism is invoked to make spindle checkpoint i DNA replication l' dmkpoint , Restriction point 2. “‘-~— — ——f-" 6—0 Flume 2-11 Control Points in the Cell Cycle. The ability of growth factors to promote passage through the restriction point illustrates how the cell cycle is regulated by external conditions. Another type of control involves checkpoint pathways that monitor conditions inside the cell and transiently halt the cell cycle at various points ifconditions are not suitable for continuing. Checkpoint pathways monitor for DNA damage, DNA replication. and chrot'nosonte attachment to the spindle, transiently halting progression through the cell cycle at various points if conditions are not suitable for continuing. certain that the chromosomes are all properly attached to the spindle. If the chromosomes are not completely attached, the cell cycle is temporarily halted at this point to allow the process to be c0mpletecl. In the absence of such a control mechanism for monitoring chromosome— to-spindle attachments, there would be no guarantee that each of the newly forming cells would receive a complete set of chromosomes. A detailed description of the spindle checkpoint and its role in the development of cancer will be provided in Chapter 10 (see Figure 10—16). A third type of checkpoint is used to prevent cells with damaged DNA from proceeding through the cell cycle. in this case, a series of DNA damage checkpoints t‘rtonitot‘ for DNA damage and halt the cell cycle at various points—including late Gt, 8, and late GE—by inhibiting different Cdk-cyclin complexes. A molecule called the p53 protein plays a central role in these checkpoint path— ways. ln the presence of damaged DNA, the p53 protein accumulates and triggers cell cycle arrest to provide time for the DNA damage to be repaired. If the damage cannot be repaired1 pS3 may also trigger cell death by opoprosis (a process to be described shortly). The ability of p53 to trigger cell cycle arrest or cell death prevents cells with damaged DNA front proliferating and passing the damage on to succeeding generations of cells. The mechanisms involved in the p153 pathway, including the way in which DNA damage is detected and the way in which pS3 triggers cell cycle arrest attd cell death, will be described in Chapter 10 (see Figure 10-5}. Cell Cycle Control Mechanisms Are Defective in Cancer Cells The preceding discussion of cell cycle control mechanisms has focused largely on the behavior of normal cells. How do these principles apply to the behavior of cancer cells, which grow and divide in art uncontrolled fashion? W’ have already seen that cancer cells often produce excessive amounts (or hyperactive versions) of growth factors. receptors. or other components of growth factor signaling pathways. Such alterations cause an excessive productitm of the Cdk—cyclins that phosphorylate the Rb protein, thereby providing an ongoing stimulus for cells to pass through the restriction point and divide. The situation is made even worse by the fact that the restriction point often fails to function properly in cancer cells. \Nhen cancer cells are grown under suboptimal conditions—for example, insufficient growth factors. high cell density, lack of anchorage, or inadequate nutrients— that would cause normal cells to become arrested at the restriction point, cancer cells continue to grow and divide without halting at the restriction point. In other words. cancer cells exhibit a ioss raj—restriction point control tinder extremely adverse conditions, such as severe nutritional deprivation, cancer cells die at random points in the cell cycle rather than arresting at the restriction point. In addition to the loss of restriction point control, cancer cells frequently exhibit defects in the checkpoint Growtl't Factors and the Cell Cycle 27 Remove both drugs I_ Add : staurosporine camptothecin [at Normal cells When cultures of normal cells are exposed to staurosporine followed by camptothecin. the staurosporine halts cells at the restriction point, thereby . preventing them from entering S phase ! and being killed by camptothecin. When the drugs are later removed, the cells again pass through the restriction point Cell cycle mwmﬁ No cell killing Cells halt at restriction point . r ‘ I-‘l‘.’E' ' and resume dividing. I lb} Cancer cells Staurosporine does not stop cancer cells in 61 because of the absence of restriction point control, so cancer cells 52 G1 exposed to staurosporine proceed into S phase and are killed by the camptothecin. Cells killed as they pass through S phase Figure 2-12 Selective Destruction ot Cancer Cells Based on the Loss of Restriction Point Control. When cells are exposed to staurosporine followed by camptothecin, cancer cells are killed but normal cells are not. IBased on tliila from X. Chen et grin]. Natl. Cancer inst. 92 (2000:: I‘J‘J‘M pathways that would otherwise respond to internal problems, such as DNA damage, by halting the cell cycle. Failures in checkpoint pathways, along with the loss of restriction point control, allow cancer cells to continue proliferating under conditions in which the cell cycle of normal cells would stop. The molecular abnormalities responsible for the restriction point and checkpoint fail- ures exhibited by cancer cells will be described in Chapters 9 and 10, which cover the genetic mutations that underlie the development of cancer. The difference between cell cycle regulation in cancer cells and normal cells can be exploited experimentally using drugs that act at different points in the cycle. For example, stnurosporine is a drug that halts the cell cycle at the restriction point, and cmrrptorhecin is a drug that kills cells in S phase by disrupting DNA synthesis. As shown in Figure 2—12. when cultures of normal cells are exposed to staurosporine followed by camptothecin, the stauro- sporine halts cells at the restriction point and thus prevents them from entering S phase and being killed by catnptothecin. [f the two drugs are later removed, the cells again pass through the restriction point and resume dividing. With cancer cells, the results are quite different. Staurosporine does not stop cancer cells in (31 because of the loss of restriction point control, so the cells proceed into S phase and are killed by camptothecin. This discovery that cancer cells can be killed by drug combinations that do not harm normal cells raises the possibility that similar strategies might eventually be devised for treating cancer patients. 28 Chapter 2 Profile of a Cancer Cell APOPTOSIS AND CELL SURVIVAL Thus far, this chapter has focused on traits that affect cancer cell proliferation by affecting the cell cycle and cell division. An unrestrained cell cycle, however, is not the only factor that contributes to the uncontrolled producv tion of tumor cells. The number of cells that accumulate in a growing tumor is determined not just by the rate at which cells divide. but also by the rate at which they die. As in the case of the cell cycle, cell death is controlled by pathways that fail to function properly in cancer cells, thereby permitting the survival of cells that would other- wise be destroyed. Apoptosis Is a Mechanism for Eliminating Unneeded or Defective Cells Cell death seems like it would he a random, uncontrolled, undesirable event. In reality, organisms possess a precisely regulated genetic program for inducing individual cells to kill themselves when appropriate. This suicide program, called apoptosis, is designed to prevent the accumulation of unneeded or defective cells that arise during embryonic development as well as later in life. For example, you might think that embryos would produce only the exact number of cells they need, but that is hardly the case. Embryos produce many extra cells that will not form part of the ﬁnal organ or tissue in which they arise. A case in point is the human hand, which starts off as a solid mass of tissue. The lingers are then carved out of the tissue by a process in which apoptosis is invoked to destroy the cells that would otherwise form a webbing between the ﬁngers. Apoptosis is also important in the newa forming brain, where extra nerve cells created during embryonic devel— opment are destroyed by apoptosis during early infancy as the ﬁnal network of nerve connections is established. Another function of apoptosis is to rid the body of defective cells. For example, cells infected with viruses often invoke apoptosis to trigger their own destruction, thereby limiting reproduction and spread of the virus. Cells with damaged DNA may also trigger apoptosis, espe— cially if the damage cannot be repaired. This ability to destroy genetically damaged cells is especially useful in helping avert the development of cancer. Apoptosis Is Carried Out by a Caspase Cascade Apoptosis is a unique type of cell death. quite different from what happens when cells are destroyed by physical injury or exposure to certain poisons. In responSe to such Cell shrinkage nonspeciﬁc damage, cells undergo necrosis, a slow type of death in which cells swell and eventually burst, spewing their contents into the surrounding tissue. Necrosis often results in an inﬂammatory reaction that can cause further cell destruction, which makes it potentially dangerous. In contrast, apoptosis kills cells in a quick and neat fashion without causing damage to surrounding tissue. The process involves a carefully orchestrated sequence of intracellular events that systematically dismantle the cell (Figure 2-13). The first observable change in a cell under— going apoptosis is Cell shrinkage. Next, small bubble—like protrusions of cytoplasm (“blebs”) start forming at the cell surface as the nucleus and other cellular structures begin to disintegrate. 'I‘he chromosomal DNA is then degraded into small pieces and the entire cell breaks apart, forming small fragments known as apoptotic bodies. Finally, the apoptotic bodies are swallowed up by neigh- boring cells called phagocytes, which are specialized for ingesting foreign matter and breaking it down into tnole— cules that can be recycled for other purposes. Cell disintegration Apoptotic Phagocytic body cell o {Caspase cascade) receptor promoting Apoptosis activated Stimulates Cytochrome c Mitochondrion proteins Hnu'e2-13 Main Steps in Apoptosis. ('l‘op) As a cell begins to undergo apoptosis, its cytoplasm shrinks and bubble-iike protrusions of cytoplasm form at the cell surface. The nucleus and other cellular structures then disintegrate and the entire cell breaks apart, forming small apoptotic bodies that are engulfed by neighboring phagocytic cells. (Bottom) The two main routes for triggering apoptosis are the external pathway and the internal pathway. :23) In the external pathway, external molecules bind to death receptors on the outer surface ofthe targeted cell, The activach death receptors then trigger the caspase cascade. -’2‘- In the internal pathway, damaged DNA triggers accumulation otthc p53 protein, which simulates the production ofcleath—promoting proteins that alter the permeability of mitochondrial membranes. This event leads to the release of a group of mitochondrial proteins, including cytochrome t‘. that activate the caspase cascade. Apoptosis and Cell Survival 29 u. Apoptosis is carried out by a series of protein- degrading enzymes known as caspases. Normally, caspases reside in cells in the form of inactive precursors called procnspnses. When a cell receives a signal to commit suicide, an initiating member of the procaspase family is converted into an active caspase. The activated caspase catalyzes the conversion of another procaspase into an active caspase, which activates yet another procaspase, and so forth. Some members of this caspase cascade destroy key cellular proteins. For example, one caspase degrades a protein involved in maintaining the structural integrity of the nucleus, and another caspase degrades a protein whose destruction releases an enzyme that causes frag— mentation of chromosomal DNA. Hence, the net effect of the caspase cascade is the activation of a series of enzymes that degrade the cell‘s main components, thereby leading to an orderly disassembly of the dying cell. Cancer Cells Are Able to Evade Apoptosis The presence of procaspases within a cell means that the cell is programmed with the seeds of its own destruction, ready to commit suicide quickly if so required. It is therefore crucial that the mechanisms employed to control caspase activation are precisely and carefully regulated and are called into play only when there is a legitimate need to destroy an unneeded or defective cell. There are two main routes for activating the caspase cascade, an external pathway and an internal pathway l[see Figure 2—13, botton-t). The external pathway is employed when a cell has been targeted for destruction by other cells in the surrounding tissue. In such cases, neighboring cells produce molecules that transmit a “death signal” by binding to death receptors present on the outer surface of the targeted cell. The activated death receptors then interact with, and trigger activation of, initiator procaspase molecules located inside the cell, thereby starting the caspase cascade. The internal pathway—a pathway that is particularly relevant to the field of cancer biology—functions mainly in the destruction of cells that have sustained extensive DNA damage. Although cells possess several mechanisms for repairing DNA damage (to be discussed shortly). in many cases it is safer to destroy cells in which there is any question about the integrity of their DNA. In this way, the potential danger posed by the proliferation of mutant cells is minimized. The pSB protein (p. 27) plays a pivotal role in the mechanism by which apoptosis is induced in cells that have sustained extensive DNA damage. The presence of damaged DNA triggers the accumulation of the p53 protein. which in turn stimulates the production of pro— teins that alter the permeability of mitochondrial membranes. The altered mitochondria then release a group of proteins, especially cytochrome c, that activate the caspase cascade and thereby cause the cell to be destroyed by apoptosis. Given that killing defective cells is one of the main functions of apoptosis, why aren1t cancer cells destroyed? After all, cancer cells fit the definition of defective cells: 30 Chapter 2 Profile of a Cancer Cell They grow in an uncontrolled fashion and. as you will learn shortly, possess DNA mutations and other chromo— somal abnormalities. The reason cancer cells are still able to survive is that they have developed ways of avoiding apoptosis. One common mechanism is that many cancer cells have mutations that disable the gene coding for p53. thereby disrupting the main internal pathway for trig— gering apoptosis. As you will learn in Chapter 10, which provides a detailed description of the pS3 pathway, muta— tions in the p53 gene are the most common genetic defect observed in human cancers. Other genes involved in apoptosis may also be altered in cancer cells. For example, the gene coding for the Bch protein, a naturally occurring inhibitor of apoptosis, is altered in some cancers in a way that causes too tnuch 8ch to be produced, thereby blocking apoptosis. DNA DAMAGE AND REPAIR As an alternative to committing suicide by apoptosis, cells with damaged DNA may try to repair the damage. DNA repair is a topic of great importance to the field of cancer biology because DNA mutations——detined as any change in DNA base sequence—play a central role in causing the uncontrolled proliferation of cancer cells. Many of the speciﬁc genes whose mutation can foster the development of cancer will be described in Chapters 9 and It]. In this chapter, we are concerned mainly with introducing the kinds of mechanisms that generate DNA mutations and seeing why cancer cells often fail to repair the damage. DNA Mutations Arise Spontaneously and in Response to Mutagens In describing how mutations arise, we first need to review a few basic principles regarding DNA structure and rep- lication. DNA molecules are constructed from two intertwined, helical strands, each consisting of a linear chain of building blocks called nucleotides. As was pointed out in Chapter 1, DNA contains four types of nucleotides, each with a different nitrogen—containing base. The four types of nucleotides are represented by the letters A, T, G, and C, which are abbreviations for the bases adenine, thyminegnonine, and cytosine, respectively {see Figure 1—8}. The nucleotide bases play two crucial roles in DNA: (1} the linear sequence of bases encodes genetic information, and (2) hydrogen bonds between the bases hold the two DNA strands together to form a double helix (Figure 2-14. left}. The hydrogen bonds that hold the double helix together only fit when they are formed between the base A in one DNA strand and T in the other, or between the base G in one DNA strand and C in the other. The interaction between A and T, or between G and C, is known as complementary base pairing. During DNA replication, the two strands of the double helix become separated and each strand functions as a template that dictates the synthesis ofa new complementary DNA strand using the base—pairing ...
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## Kleinsmith_ch2 - FEATURES OF CANCER CELL PROLIFERATION The...

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