cancer research€¦ · autoradiography with tritium-labeled precursors of nucleic acids and...

VOLUME 25 JUNE 1965 NUMBER 5

sequence of events leading from one mitotic division to thenext, and to understand the way in which knowledge ofthese events helps to clarify some of the problems relatedto kinetics of cellular proliferation and tumor growth.

THE CELL CYCLE

Our knowledge of the cell cycle has been obtainedmostly by the use of high-resolution autoradiography(20, 63). The interested reader is referred to the literaturefor a detailed description of the technic of high-resolutionautoradiography (14, 61, 62, 107, 124, 140, 169, 225), andof the various radioactive precursors that are most frequently used to investigate the metabolic activities of cells(3, 4, 6, 7, 13, 14, 44, 61, 62, 64, 74, 131, 167, 194, 216, 218,247, 256). The most commonly used nrecursor is tritium

For students of cellular proliferation, the most important event in the life of a cell has always been mitosis, inits classic stages from prophase to telophase (248). However, in recent years it has been found that the long interval between 2 successive mitoses, called the interphase,included a series of metabolic events that are of importancein the over-all process of cell division. The orderly sequence of these metabolic activities, from midpoint ofmitosis to midpoint of a successive mitosis, constituteswhat is called the cell cycle. The object of this review isto present a general outline of the cell cycle, to describe the

1 TJSPHS Research Career Development awardee (5-K3-GM15, 465-05).

ReceivedforpublicationSeptember 2, 1964;revisedJanuary 15, 1965.

581 This One

CANCER RESEARCH

The Relationship of the Cell Cycle to Tumor Growth andControl of Cell Division: A Review

RENArO BASERGA'

(Department of Pathology, Northwestern University Medical School, Chicago, Illinois)

SUMMARY

Metabolic activities of cells during interphase can be studied by high-resolutionautoradiography with tritium-labeled precursors of nucleic acids and proteins. Aftercompletion of mitosis, offspring of the parent cell may divide again, or may differentiateand die without further division. Dividing cells go through a series of metabolicchanges which constitute the cell cycle. The cell cycle is subdivided into 4 distinctphases: (a) a postmitotic phase called G,, during which the cell synthesizes ribonucleic acid (RNA) and proteins; (b) an S phase, during which the amount of deoxyribonuleic acid (DNA) is duplicated while protein and RNA syntheses continue;(c) a postsynthetic phase, called G2, characterized by no net synthesis of DNA and continued RNA and protein synthesis as in postmitotic phase; and (d) mitosis, extendingfrom beginning of prophase to completion of telophase during which there is no DNAsynthesis, protein synthesis is reduced to a minimum, and RNA synthesis is limited toearly prophase and late telophase. The length of the cell cycle and its 4 phases can beobtained by appropriate experimentation with labeled thymidine, a precursor of DNA,andhigh-resolutionautoradiography. Although thelengthof the ceilcycle variesin different kinds of cells, it is shorter in certain cells of the adult animal than in some of thefastest growing tumors. Tumor growth, therefore, must involve other kinetic parameters besides speed of cell proliferation. Since these parameters are related to controlof cell division and since a cell in DNA synthesis is generally a cell that has alreadytaken the decision to divide, the various factors involved in initiation of DNA biosynthesis are of particular interest in any attempt to explain the mechanism of tumorgrowth. It is possible that the initiation of DNA biosynthesis may involve DNAitself, and may be mediated through synthesis of specific RNA and enzymes.

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582 Cancer Research Vol. 25, June 1965

labeled thymidine (thymidine-3H), a deoxynucleosidewhich is incorporated exclusively into DNA (69, 200).Since thymidine and its degradation products are solublein ordinary fixatives, in which DNA is insoluble, anyradioactivity found in fixed tissues or cells after exposureto radioactive thymidine is due to newly synthesized DNA(5, 215), a conclusion confirmed by histochemical studies(4, 14). In addition, thymidine, when injected into intactanimals, is either promptly incorporated into DNA orcatabolized to nonutilizable products (189, 255). Thetime during which injected thymidine is available forincorporation into DNA is about 30 mm (119, 135), sothat for all practical purposes, an in vivo exposure tothymidine results in pulse labeling The cell cycle wasdiscovered by Howard and Pelc (104). Numerous modifications have been introduced since their original experiment, but the basic model remains the same. Let us suppose an asynchronous population of cells, such as thelining epithelium in the crypts of the small intestine ofmice. When thymidine.-H' is injected into mice, cellsthat are synthesizing DNA at the time of injection take upthymidine and become labeled. An autoradiograph of asection of small intestine, 30 mm after injection of thymidine, discloses 3 types of epithelial cells in the crypts:cells in mitosis (all unlabeled), unlabeled interphase cells,and labeled interphase cells. The 1st bit of information wehave obtained is that DNA is synthesized during interphase and not during mitosis, since all cells in mitosis areunlabeled. But because of the short period of time duringwhich thymidine is available, and the fact that DNA isstable (21, 97, 116, 150, 202), we can now follow the fate ofcells that were in DNA synthesis at the time of injection

by taking samples at different intervals after injection ofthymidine. For 30 min or so after exposure to thymidine@Hall mitoses are unlabeled, indicating that there is a

measurable period before mitosis during which cells do notsynthesize DNA. After 30 mm, the percentage of labeledmitoses increases and the interval between administrationof thymidine and the time at which 50 % of mitoses arelabeled gives the duration of the premitotic phase, calledthe G2 phase (104, 128) during which no DNA is synthesized. As more and more cells that were in DNA synthesis at the time of thymidine injection enter mitosis, thepercentage of labeled mitoses increases until 100 % of themitoses are labeled (Chart 1). After a while, the percentage of labeled mitoses decreases. The interval between the two 50 % points on the ascending and descendinglimbs of the curve representing percentage of labeledmitoses (Chart 1) gives the duration of the phase duringwhich DNA is synthesized, called the S phase (104, 128,197, 198). The length of the entire cell cycle can beestimated on the basis of the time elapsing between 2 successive peaks of labeled mitoses (70, 219). In Chart 1, theinterval between the midpoints of the 1st and 2nd peaks is12 hr and the length of the cell cycle is then estimated at12 hr. The duration of G2 is 1 hr, and the length of S phase,7 hr. Since mitoses usually last less than 1 hr (71, 99,139, 250) another phase must be postulated to complete the12 hr of the cell cycle. The existence of this phase is alsoindicated by the decrease in percentage of labeled mitosesafter the 8th hr, which suggests a period, preceding DNAsynthesis, during which no DNA is synthesized. Theduration of this period, called the G1 phase, can be ob

0Lu-JLu

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Li

0I-0I-.

zLu0

LuQ.

CHART 1.â€”Percentage of labeled mitoses in the lining epithelium of thecrypts of the small intestine of mice at various intervals after a single injection of â€˜H-thymidine (see explanation in the text). Reproduced, with permission, from the paper by Lesher et at. (137). The mice were injected at 0 timewith 50 pc of thymidine-'H. In this particular experiment, the cell cycle wasstudied in the intestine of chronically irradiated animals. One of thesegroups was chosen for the present illustration, because the amount of variation at the various points was remarkably low. However, the curve for norma! mice was essentially similar.

I00

8 12TIME IN HOURS AFTER INJECTION

OF TRITIATED THYMIDINE



BASERGAâ€”Cell Cycle and Tumor Growth 583

of the label, increases for a time equal to T8 and thenreaches a plateau. A variant of this approach, describedby Stanners and Till in tissue cultures (228), has beenapplied in vivo by Koburg (123). T, can also be calculatedby exposing cells to 2 different pulses, one of thymidineâ€˜4Cand the other of thymidine-3H (16, 186, 252). Cellslabeled with thymidine-'4C only can be distinguished fromcells labeled with thymidine-@H only or with both isotopesby 2-emulsion autoradiography (17), and the length of theS phase can be calculated from appropriate equations(252). Although each of these methods is useful inparticular situations, the one described in Chart 1 muststill be considered the most accurate and the method ofchoice whenever feasible (197).

The length of the S phase in mammalian cells is oftenvery close to 8 hr (42, 159, 175, 228, 254). However, thenumber of exceptions to this rule has been rapidly increasing in the past few years. Certain near-tetraploid ascitestumors of mouse have a T8 of 11â€”12hr (8, 55, 102), andother instances of T8 of unusual length have been describedin cells of germinal centers of rat spleen, 4.5 hr (66); in dogmyeloblasts, 5 hr (179) ; in epithelial cells of human colon,9 hr (145) ; in transplantable mammary tumors of mouse,10 hr (159); in epithelial cells of mouse forestomach, 13.5hr (253); in mouse spermatogonia, 14 hr (163) ; in alveolarcells of mouse mammary gland, 20 hr (35); and in epithehal cells of mouse ear epidermis, 30 hr (220). T@may evenvary in the same tissue under different conditions, as inthe intestinal epithelium of mice, where T@in chronicallyirradiated mice is 1 hr shorter than in controls (137). InEhrlich ascites cells T8 is shorter in diploid than in neartetraploid lines (55). Actually, in the same hypotetraploid line, both T8 and T@may vary with the sex of therecipient mouse. In the Ehrlich tumor described byBaserga and co-workers, T8 and T@last, respectively, 11and 18 hr in male mice (8) and 17 and 36 hr in females (9).Thus, the original assumption of the constancy of the Sphase in mammalian cells is no longer tenable.

DNA synthesis is only one of several synthetic activitiesthat take place during the S phase. Cells synthesizeRNA throughout the entire interphase, as evidenced bythe fact that nearly all interphase cells are labeled by abrief exposure to a radioactive RNA precursor (94, 237).In the ciliated protozoan Euplotes, Prescott has shown thatDNA and RNA syntheses are mutually exclusive, asevidenced by the lack of uptake of RNA precursors inregions of nuclei at the time of DNA replication (193, 195).It is also known that RNA polymerase is more active whenprimed by native nonreplicating DNA than by singlestranded DNA (73, 111). However, in mammalian cells,because of asynchrony in DNA replication (143), the rateof uptake of RNA precursors is substantially the same incells in DNA synthesis and in other cells in interphase (6).

The rate of protein synthesis, as measured by uptake oflabeled amino acids, is increased during the S phase. Thiswas shown directly in protozoa by Prescott (192), inonion root meristem by Olszewska (173), and in mammalian cells by Baserga (7) and Baserga and Kisieleski(13). This conclusion is also supported by the findings ofBloch and Godman (29) and Dc (54) on histone synthesis

(A) and by those of Lipkin et al. (144) that the uptake of radio

MI'FO$I@

CELL$

NON -DIVIDINGCuLL

CHART 2.â€”The life cycle of cells. At completion of mitosis,daughter cells may go through another life cycle (G1, S and G2),or become nondividing cells, destined to die without any furtherdivision.

tained by subtracting from the entire length of the cellcycle the duration of G2 + S + mitosis.

The cell cycle (104, 128, 197) may then be defined as theinterval between midpoint of mitosis in the parent celland midpoint of the subsequent mitosis in the daughtercell (Chart 2). If one of the sister cells is destined not todivide again, it leaves the cell cycle and becomes a nondividing cell that may eventually die or persist for theentire life of the organism without dividing (Chart 2).The lining epithelium of the small intestine can be used toillustrate an admixture of dividing and nondividing cells,although similar admixtures occur in other tissues (181).DNA synthesis and cell division occur only in the crypts(136, 198). From the crypts, cells migrate to the villus,where they become nondividing cells, destined to be shedinto the intestinal lumen without any further mitotic

division.

THE 4 PHASES OF THE CELL CYCLE2

S phase.â€”Wehave seen that DNA synthesis takes placein a discrete period of interphase, called the S phase (104,236, 246). There are several ways of determining theaverage length of the S phase, T.. The most commonlyused is the one described above in Chart 1 (70). Anotherway of determining T8 is by continous labeling (251). Ifcells are constantly exposed to tritiated thymidine, as ininfusion technics (180) or in tissue cultures (228), thegrain count per cell, that is, the autoradiographic intensity

2 We have adopted the nomenclature and definitions that were

agreed upon among the participants to the Guinness Symposiumon Cell Proliferation (197). Besides the definitions given above ofthe cell cycle and its phases or compartments (5, G1, G,, M),these include the following conventions : N@means the number ofcells in a compartment, and therefore N8 is the number of cellsin the S phase, N@ the total number of cells in the population,etc. Similarly, T@is the transit time or the time elapsed betweenentering and leaving a compartment : e.g., T@is the length of cellcycle, T. the length of the S phase, etc. The thymidine index,I, is defined here as the percentage of cells labeled within a30-mm interval after a single injection of radioactive thymidine(13).In theabovenotation:

lOON,Iâ€” N@




active leucine by the small intestine is higher in crypt cellsthan in villus cells, where no DNA synthesis occurs.However, DNA and protein synthesis may proceedindependently from each other, as shown by Maaloe (147)in bacteria, and by Rueckert and Mueller (207) andRusconi (208) in mammalian cells.

Postsynthetic phase.â€”In this phase, the G2 phase, cellssynthesize RNA and proteins but not DNA (6, 7, 94, 170,192, 237). The length of the postsynthetic phase is fairlyconstant in mammalian cells, ranging from@ to 1@hr (67,175, 197, 228). There are exceptions, like cells of the earepidermis of mouse (220), pre-enamel cells of the developing enamel organ in teeth of young rabbits, in which theG2 phase lasts from 6 to 9 hr (229), some near-tetraploidascites tumors of mouse with a G2 phase of about 6 hr (8,55), and mouse spermatogonia, that have a variable G2time (163).

Mitosis.â€”The duration of mitosis has been estimated tovary in different tissues from 30 mm to 2.5 hr (71, 99, 139,201, 250). It may be mentioned briefly that, duringmitosis, protein synthesis reaches a minimum (7, 13) andRNA synthesis is limited to early prophase and latetelophase (51, 194). The end result of mitosis is reductionof the amount of DNA per cell to the diploid value (236)characteristic of the species (30).

Postmitotic phase.â€”At completion of mitosis, the cellenters the G, phase. Of the 4 phases of the cell cycle, thisis the most variable in length (67, 159, 238). It is veryshort, practically undetectable by ordinary methods, in theslime mold Physarum polycephalum (170), in the sea urchinembryo (153), in Ehrlich ascites cells growing in theperitoneal cavity of male mice (8), and in pre-enamel cellsof young rabbits (229), while in other mammalian cells itmay last from a few hours to several months (181). It isalso the phase in which most cells with a long cycle timestop for an indefinite time as evidenced by the followingfindings : (a) in tissue cultures the S and G2 phases areconstant and variations in length of the cell cycle dependupon variations in length of the G1 period (224, 238); (b)in adrenal gland of rats, the length of the cell cycle variesfrom 80 hr in cells of the glomerulosa to about 250 hr inthose of the outer fasciculata. Since the length of 5, G2,and mitosis appears to be the same, the phase that variesin length must be the G1 (67); (c) in regenerating liver, thecurve for percentage of hepatocytes in DNA synthesis atdifferent intervals after partial hepatectomy precedes byabout 6â€”8hr, both in initial elevation and peak incidence,the curve for mitotic index (87, 138). There are, however,exceptions. Gelfant (75) was the first to suggest that somecells of mouse ear epidermis may be arrested, not in G1,butin the G2 phase, from which they entered mitosis after asuitable stimulation. Cameron and Cleffmann, takingadvantage of the fact that cell proliferation, depressed bystarvation, can be stimulated by refeeding (133, 164), havedetermined variations in mitotic and thymidine indices ofstarved chickens with time after refeeding. These authorswere able to show that practically all epithelial cells of theduodenum were resting in G1, while a sizable portion ofesophageal cells were in G2, from which they proceededdirectly to mitosis (43). Starkey (229) also found that, inrabbit teeth, enamel is elaborated by cells that have

synthesized DNA without proceeding to mitosis, and asimilar situation has been reported by Owen and MacPherson (174) in rabbit osteocytes. These cells arrestedin the G2 phase must contain at least twice the amount ofDNA of interphase diploid cells, that is, they are polyploidcells, a condition that is frequently found in liver cells andother tissues of rodents (39, 105, 235, 236) and is the rulein myocardial cells of man (209). Despite these exceptions, in most instances, cells rest in the G1 period of thecycle (165).

Cellular sites of nucleic acid and protein syntheses.â€”Thereis general agreement that DNA is synthesized in thenucleus, with the exception of DNA viruses, which canreplicate in the cytoplasm (112), and possibly of the DNAfraction located in mitochondria (76). It is also agreedthat proteins are synthesized in the nucleus as well as inthe cytoplasm (14, 172, 247), but there is still some uncertainty as to the sites of synthesis of RNA. This subjecthas been reviewed recently by Graham and Rake (84).Briefly, we can say that the bulk of evidence indicates thatRNA is synthesized in the nucleus, from which it migratesto the cytoplasm (83, 223). A large portion of RNA has ahigh turnover rate and breaks down rapidly after beingsynthesized (95). Soluble RNA and ribosomal RNA arealso synthesized in the nucleus (40, 45, 78), ribosomalRNA actually in the nucleolus (36, 154, 183, 184), andthen transferred, at least in part, to the cytoplasm (223).

THE STEADY STATE SYSTEM

Many tissues of the adult animal are in a steady state inrespect to the total number of cells. This means that notonly does the total number of cells remain constant, butthe number of cells in each compartment also remainsconstant. A good example of a steady state system is thelining epithelium of the mucosa of the small intestine (132,198). The epithelial cells that line the intestinal mucosabelong to 2 distinct compartments : the crypt and thevillus. The crypt is where cell division occurs (28) and itis called the progenitor compartment. The villus is thefunctional compartment, where cells do not divide. Whilecells from the crypt migrate to the vifius, cells almostnever return from the functional into the progenitorcompartment (198). Each time a cell in the crypt dividesinto 2 daughter cells, another cell migrates from the cryptinto the villus. Similarly, in the villus, for every cellentering it from the crypt, another cell leaves at the tipand is shed into the intestinal lumen, where it dies (132,136, 198). If the number of cells remains constant, itfollows that 1 of the 2 cells produced by cell division isdestined to die without dividing again. It would betempting to speculate that, of 2 sister cells produced byeach division, 1 divides again and the other migrates to thevillus and dies. This is not necessarily true and it ispossible that, in some instances, both sister cells divideagain, while in other instances both sister cells leave thecrypt without any further division. Leblond (130)inclines to the opinion that, at least in esophagus andintestinal epithelium, the decision as to whether a proliferating cell will differentiate or continue to divide takesplace during interphase and depends on local environment.

The various parameters of cell division and migration in



BASERGAâ€”Ce11 Cycle and Tumor Growth 585

the lining epitheium of the intestine have been describedin detail, in mouse by Quastler and Sherman (198) andLesher et al. (136), in rat by Leblond and Stevens (132),and in man by Lipkin et al. (145), and are substantiallysimilar in various species. Analogous systems are foundelsewhere in the body, for instance in squamous epitheliumlining the skin and mucosae of internal cavities (182).Steady state systems like bone (113) and bone marrow (32,177, 180) are rather more complicated, but they arealso characterized by the fact that the total number ofcells remains constant, with cell production balanced bycell loss. In the rat, several cell populations, like those ofthe kidney, pancreas, or skeletal muscle, that in man areconsidered in a steady state system because the weightsof the organs plateau in adult life (199), are in fact slowlyexpanding until 1 year of age (60). During this time, theonly tissue that shows no increase in cell number is cerebellum (60). After 1 year of age, however, these expandlug cell populations also become steady state systems (130).

LENGTH OF CELL CYCLE

We have defined T., as the mean transit time around theentire cell cycle, that is, the time elapsed between the midpoints of 2 successive mitoses. Sisken and Kinosita (224)have determined the length of the cell cycle of individualcells in tissue culture by time-lapse microcinematography.These authors followed individual cells in a colony ofhuman amnion cells from one anaphase to the next andfound T@= 21.6 hr Â± 0.5. In vivo measurements ofindividual cells are not available yet, and thus, T@usuallygives the mean transit time for a cell population withoutany knowledge of individual variations. Variations inlength of the cell cycle by individual cells may make T@rather meaningless. For instance, if a population of cellsconsists of 2 distinct sub-populations, one with a long cellcycle and a second one with a short cell cycle, the meancycle time T@may mean very little. Such populationshave been described in the forestomach of mice by Wolfsberg (253) and in macrophages of pulmonary alveoli ofmice by Shorter et al. (222). Even in a more homogeneouspopulation, individual cycle times of cells may varygreatly about the mean although the direct measurementsof Sisken and Kinosita (224) indicate small variations, apossibility confirmed by the findings that the length of T@may be measured by the interval between 2 successivepeaks of labeled mitoses (70), and that in bacteria thecycle time has a normal distribution (126). However, inmammary tumors of mice, the 2nd and 3rd waves oflabeled mitoses are lower than the 1st peak, and the cellshave completely desynchronized by the 4th generation.Together with the damped 2nd wave of labeled mitosesdescribed by Johnson in a transplantable mouse sarcoma(108), they indicate a considerable amount of variance incycle times of individual cells, at least in some populationsof cells. However, until methods become available fordetermining T, of individual cells in vivo, one should notreject as meaningless the measurements that can be obtamed by the methods to be described below.

The classicmethodsforstudyingsomeof the parametersof cell proliferation have been based on the use of colchicine (24, 129, 231), and a combined method for deter

mining T@,using both thymidine-3H and colchicine, hasbeen introduced recently by Van't Hof and Ying (244)and applied to root meristems of Pisum satwum. Amongthe methods using only high-resolution autoradiographyand labeled DNA precursors, the one illustrated in Chart1 probably represents the most straightforward way ofdetermining T@. Unfortunately, it is not always feasiblebecause, as mentioned above, some cell populationsdesynchronize in 1 generation and the 2nd wave of labeledmitoses fails to materialize (108). The possibility existsthat failure of the 2nd wave of labeled mitoses to materialize may be due to a statistical error inherent in a smallnumber of observations. For instance, in his originaldescription of 28 mammary tumors of C3H mice, Mendelsohn (155) could not detect a 2nd wave of labeled mitoses,but when an additional 92 tumors were investigated, hewas able to recognize that the 1st wave of labeled mitoseswas followed by 2 other recognizable and equally spacedwaves (158). It is therefore conceivable that the methoddescribed by Fry â‚¬1al. (70) may turn out to have a widerapplication than presently allowed.

Another way of determining T@is based on the fact thatT, can be determined independently in an accurate way(see above), and that the relation between T, and T@canbe expressed by the equation:

Na/Nc = T,/TC (B)

where N, is the number of cells labeled by a pulse-exposureto thymidine-3H and N@is the total number of cells in thepopulation. This equation is valid in an ideal system(197), but, in practice, it can be used in only a few selectedinstances. Apart from the fact that N, may undergodiurnal fluctuations (68, 185), a serious shortcoming ofEquation B is that it requires the assumption that all cellsin the population participate in the proliferative process.Actually, most proliferative compartments contain admixtures of cells which are not going to reproduce. Theratio NI/NC will then be less than the ratio NI/NP, whereN@ is the total number of cells in the proliferative cycle(157). The result is an overestimate of the length of thecell cycle. For instance, T, in the epitheial cells liningthe small intestine of mice is 7 hr. The ratio N,/N@ isabout 0.065 if determined on all epithelial cells lining theintestinal epithelium, that is, epithelial cells of the cryptsas well as nondividing cells of the vffli. If only cryptepithelial cells are counted, the ratio is 0.45. By rearranging and substituting in Equation B, one would obtain2 values of Tc of 107 and 15 hr, respectively, a substantialdifference. When using Equation B it is thereforeimportant to know N,,, defined by Mendelsohn (155, 157)as the Growth Fraction, or the fraction of cells in the totalpopulation that participate in the proliferative process.Since N, = pNc (where p is the fraction of proliferatingcells), the problem is how to determine p in a cell population in vivo. One way around it is to obtain 7.'@from theequation:

Tc M*/M(C)

where M* is the number of labeled mitoses and M thetotal number of mitoses (157). The equation requires an




independent determination of T,, followed by a determination of M*/M after the labeled cells have desynchronized. A difficulty with this method is that if onewaits too long for cells to become properly desynchronized,the concomitant dilution of the label makes recognition oflabeled cells increasingly uncertain. On the other hand,if cells desynchronize rapidly, the T@obtained will represent a mean to which individual cells confirm verypoorly (157). An alternative is to determine the fractionof proliferating cells, p, from the number of cells that arelabeled by continuous exposure to thymidine-'H providedonly the progenitor compartment is taken into consideration. In tissue cultures, p can be determined by incubating cells with thymidine-3H and observing the number ofcells that become labeled over an extended period of time(228). An in vivo modification was applied by Basergaet al. (15) to Ehrlich ascites cells growing in lungs of mice.Tumor-bearing animals were injected every 4 hr withthymidine-3H, and since all cells were found to be labeledafter 6 injections, it was concluded that all cells participated in the proliferative process, that is p = 1. Continous incubation or repeated injections can be replacedby continous infusion of thymidine-3H (156). When thelatter method was used in mammary tumors of C3Hmice, a number of morphologically intact tumor cells remained unlabeled even after infusions as long as 10 days.This indicated that p in these tumors was considerablybelow 1. Precise measurements showed p = 0.18 (158).

Another method for determining Tc is based on thedilution of the label. In a population of labeled cells inwhich all cells divide, the specific activity of DNA decreases in direct proportion to the number of cell divisions(150, 202). In autoradiographs, the decrease in specificactivity of DNA can be followed by determining the meangrain count of cells pulse-labeled with thymidine-'H.Using this principle, Baserga et at. (18) have proposed amethod based on the distribution of grain counts thatgives at the same time p and T@. The method requires 2determinations of the mean and distribution of the graincounts of a cell population at different intervals after asingle injection of thymidine-3H. The 1st determinationmust be taken after the labeled cells have undergone the1st division following exposure to thymidine. From thegrain count mean and variance at time t0. and at timeto + @It, it is possible to obtain the proportion of cellsthat have not divided, a, that have divided once, b, andthat have divided twice, c, in the interval L@t(18).

The length of the cell cycle, T@, of the proliferatingsubpopulation is then given by:

I@tT,=@â€”@-

The method requires that the interval @tbe > T@< [email protected] gives a good first approximation of both p (in this case,1 â€”a) and T@but it again requires a rather rapid desynchronization of labeled cells. A more elaborate statisticalmethod has been proposed recently by Tyler and Baserga(241), but it would go beyond the purpose of the presentdiscussion.

In conclusion, while there are several methods that canbe used to determine T@,each of them has limitations that

restrict its use to selected situations. In practice it isoften difficult to differentiate nonproliferating cells fromcells with a very long cycle, the G0 cells of Patt andQuastler (181). For instance, there are cells with a longcell cycle which are still capable of DNA synthesis andwhich, under proper conditions may shorten the length ofthe cycle. A remarkable example of this kind is the liver,ordinarily a rather quiescent tissue with a low thymidineindex (11, 58, 162, 221), which can markedly increase itscapacity for DNA synthesis during the regenerative phasethat follows partial hepatectomy (37, 38, 87, 138). Epithelial cells of the anterior surface of the crystalline lens(92), basal cells of the epidermis (98), smooth muscle cellsof arteries and arterioles (50), regenerating skeletal musclecells (27, 120), and kidney cells (100) are also cells knownto respond to an appropriate stimulus with an increase inthe number of cells synthesizing DNA and a shortening ofthe cycle time. Other cells, like keratinizing epithelialcells of the upper layer of the epidermis, various neurones,cells of the nervous layer of the retina, and polymorphonuclear leukocytes are not known to be capable of takingup thymidine, at least in the adult mouse (58, 162, 221),and should be considered as cells that have left the cycle.

Because of the limitations mentioned above, most of themeasurements of T@reported in the literature must beconsidered with caution. A large majority of thesemeasurements are simply based on determinations of Iand T, and the use of equation B, with its attendant inconveniences. In only a few instances has T@actuallybeen determined, either by the method of Fry et al. (70)or by using N, to modify equation B. Some of thesemeasurements and estimates in man and mice are summarized in Table 1. The figures given for human tissuesare all approximate estimates but in mice we have somemeasurements that can be accepted with greater confidence. For instance, the length of the cell cycle in theepithelium of the small intestine has been determined bythe method of Fry et at. (70), and that of the Ehrlichascites tumor by using Equation B, since, in this tumor inthe exponential phase, p = 1 (146). It will be noted thatthe length of the cell cycle is shorter in the non-growingnormal adult tissue than in the rapidly growing Ehrlichtumor.

KINETICS OF TUMOR GROWTH

The simplest equation that describes the growth of atumor is of the exponential type y = bx, and it seems tofit best some ascites tumors. Patt and Blackford (178)used it to describe the 1st week of growth of Krebs ascitestumor, Maruyama (150) fitted it to the growth of LSA

(D) ascites tumor, and Baserga (9) found an exponentialpattern of growth in Ehrlich ascites tumor growing in theperitoneal cavity of mice, regardless of the method used toestimate growth, e.g., number of tumor cells, dry weight oftumor cells, or total amount of tumor DNA. In all thesecases, however, the curves flatten after the 1st week or soof growth, usually when the total number of tumor cellsper mouse reaches a figure of 500â€”600million cells (9, 178).Probably for this reason, Klein and Revesz (121) thoughtthat the growth curve of Ehrlich ascites carcinoma wasbetter expressed in terms of the cube roots of tumor cell



TissueEstimatedlengthReferenceMAN:Epidermis1.5

mo110Polychromatophilnormoblasts15â€”18hr179Epithelium

of large intestine25hr145Epitheliumof large intestine53hr46Carcinomaof colon, metastases27hr10Leukemic

blast cells50â€”80hr118Carcinomaof stomach66hr10Carcinomaof colon, metastasis75hr10Multiple

myeloma cells2â€”6days117Carcinomaof liver10days10Glioblastoma

multiforme1mo109Ovariancystadenoma1.5mo110Ovarian

carcinoma1.5mo13Carcinomaof breast3mo110Leiomyoma

of spermatic cord5.5mo110MOUSE:Epithelium

of small bowel11.6hr136Epitheliumof epidermis150hr57Bronchial

epithelium18days122Trophoblastcells, 12th-day pla 15hr42centaEhrlich

ascites tumor18hr8Skin

cancer32hr57Melanoma(transplanted)2.5days26Breast

cancer1â€”3.5 days159


TABLE 1

ESTIMATED LENGTH OF THE CELL CYCLE IN DIFFERENT

TYPES OF CELLS IN Vivo

dN ln 2dt Tc

(F)

Since both T@and N can be determined experimentally,dN/dt can be obtained by substitution. If T@is unknown,dN/dt can be calculated from the equation given byStanners and Till (228).

These manipulations are valid in an exponentiallygrowing population, but since only ascites tumors in theirinitial stages grow exponentially, exponential equations areof limited value in describing the actual growth of a solidtumor (157). Mendelsohn (158) has reiterated that solidtumors, like mammary tumors of mice and human tumors,do not conform to an exponential pattern of growth, andthat different equations must be used to describe thegrowth pattern of tumors in which the nonproliferatingsubpopulation of tumor cells constitutes a substantialpart of the total cell population. A plausible model forsuch a mixed population is the stochastic model of cellproliferation and differentiation proposed by Till et al.(239), based on a â€œbirth-and-deathâ€• process, for details ofwhich we must refer the reader to the original paper. Thesituation is further complicated by the fact that a tumoris not a closed system in which there is no cell loss from thecompartment. To begin with, necrosis, that is, cell death,is a frequent occurrence, especially in solid tumors. Inaddition to cell loss because of necrosis, of individual cellsor of entire portions of the tumor, cell loss may occur because of migration of cells out of the main proliferativecompartment. Metastases are instances of cell migration,although the number of metastasizing cells is usuallynegligible (47, 257) and secondary colonies established bytumor cell emboli can be considered as independentcompartments. More important is migration of cells insurrounding tissues, which is of such magnitude in someascites tumors as to produce an actual decrease in totalnumber of cells in late stages of growth, although some cellproliferation is still taking place (9, 59).

The growth pattern of a tumor is important not only inany attempt to understand how a tumor grows, but alsobecause the kinetics of the system require corrections inthe equations that we have found useful for measuring T@in a steady state system. For the purpose of the presentdiscussion, we shall limit ourselves to corrections necessaryin case of exponential growth. Although this is not alwayscorrect, as mentioned above, we should remember that theerror may be of little value in comparative studies. Forinstance, Reiskin and Mendelsohn (201), using an equation valid for linear growth, estimated the cell cycle timeof induced epithelial tumors of the hamster pouch at 20.7hr, and assuming an exponential growth at 17.5 hr. Thedifference may seem substantial but it becomes much lessimpressive when it is compared to the cell cycle time of the

(E) normal counterpart,the squamousepitheliumof thehamster pouch, estimated at 142 hr.

Several corrections have been proposed in the case ofexponentially growing populations (59, 108, 135), most ofwhich are useful in particular situations. We have foundconvenient, in studying the Ehrlich ascites tumor, toadopt the correction proposed by Johnson (108), thegeneral statement of which is expressed in the equation:

numbers. If a generalization must be drawn from thegrowth curves proposed for ascites tumors, it is perhapsthe conclusion reached by Patt and Blackford (178) thatthe initial growth pattern may be approximated by anexponential fit and a portion of the curve by a cube roottransform, and that neither the exponential nor the cuberoot transformation accounts for the entire growth pattern.

However, even if we accept an exponential equation todescribe the initial pattern of growth of some ascitestumors, a cube root equation seems to apply best to thegrowth of solid tumors (152, 213, 214), especially those inwhich, because of extensive central necrosis, growthdepends mainly on the peripheral cellular layers of thetumor nodule. Actually, Mayneord (152) believed thatthe cubic root pattern of growth transforms into an exponential one when necrosis is reduced to a minimum andall cells participate in the proliferating process. Bothequations have been summarized by Mendelsohn (157) ina single differential equation:

@ =

where N is the number of cells, t is time, and K a growthconstant. The expression dN/dt is the growth rate of acell population at any instant. In the case of an exponentially growing population, b in Equation E is equalto 1, and K = X = in 2/T, where T is the doubling time(108). Equation E may now be written:



tion . . . should be discarded as a general concept of thenature of leukemia,â€•and, more recently, in the findings ofPost and Hoffman (187, 188). The picture may be moredifficult to comprehend than the traditional uncomplicatedpicture of tumor cells proliferating at full speed, but it isprobably closer to the truth and opens new horizons for aproper design of chemotherapeutic or radiotherapeuticprocedures.

CONTROL OF DNA BIOSYNTHESIS

An increased production of cells, caused by eithershortened length of the cell cycle or increased size of the

(H) proliferating pool, ultimately means an alteration in thecontrol of cell division. In previous sections, we have seenthat a cell that has synthesized DNA is generally a cellthat has already made the decision to divide. Control ofcell division (233, 234) then becomes control of the initiation of DNA biosynthesis. Factors that may intervene intemporal regulation of DNA biosynthesis (127) areconveniently grouped in : (a) factors that may control DNAsynthesis through the availability of DNA precursors andrelated enzymes; (b) the physical state of the DNAmolecule; and (c) regulatory mechanisms related to thesynthesis of RNA and proteins.

DNA synthesis in vitro requires primer DNA, the 4-component deoxynucleotides, and the enzyme, DNApolymerase (125), and it is reasonable to assume that theavailability of precursors and enzyme is a necessary condition for the synthesis of new DNA. However, it may wellbe that both precursors and enzymes are manufactured inresponse to a message issued by the cell after it has takenthe decision to synthesize DNA. Several kinds ofevidence suggests that the concentration of DNA precursors and related enzymes may be determinant in theinitiation of DNA synthesis: (a) deoxynucleotides, a!though present in small amounts in tissues of the adultanimal (210), are found in increased amounts in proliferating tissues, like regenerating liver (212) and tumor tissue(206, 211); (l@) pyrimidines, purines, and nucleotidesdecrease the helix-coil transition temperature of thymusDNA, that is, they favor the transition of the DNAmolecule from the 2-stranded helical form to the singlestranded coiled form (240); (c) the concentration ofenzymes concerned with the phosphorylation of thymidineand the polymerization of deoxynucleotides, such asthymidine kinase, thymidylate synthetase, and DNApolymerase, increases in proliferating tissues (53, 86, 106,171). However, in regenerating liver, the activity of theseenzymes reaches a maximum after the rate of DNA synthesis has begun to decline (31, 101). Similarly, incultures of L cells, DNA polymerase activity increaseswhen DNA synthesis is inhibited and decreases whenDNA synthesis is allowed to proceed (82). An interestingalternative has been suggested by Kielley (115), who foundthat thymidylate kinase activity was about equal innormal mouse liver and in hepatomas, but in the formerthe activity was particle-bound and did not become evident until after disruption of the cellular constituents. Ifone had to evaluate the present evidence on the concentration of enzymes and precursors in relation to DNA synthesis, one would be tempted to conclude that while these


convenient, in studying the Ehrlich ascites tumor, adoptionof the correction proposed by Johnson (108), the generalstatement of which is the equation:

(i_t@) TI@=ln2â€¢2 T@ .@ (G)

where I@is the fraction of the total population in a givenphase, T1 the phase duration, and t the average age of cellsin that phase, expressed in terms of T@. For instance, todetermine the fraction of the cell cycle that the Ehrlichascites cells spend in S phase, the equation becomes:

I = in 2 . 2@''@ .

where I is the thymidine index and T, the S-transit time(9, 108). The equation can be solved for T,/T@to obtainthe fraction of the cell cycle that the Ehrlich cells spend inDNA synthesis. If the duration of the S phase is known(see above), T@can be obtained, and conversely, T, can beestimated if T@is known.

Comparison between tumors and normal tissves.â€”As canbe seen from Table 1, the cell cycle time of the fastestgrowing experimental tumors is actually longer than thecycle time of some normal tissues. In only 3 cases hasthe cycle time of tumor cells been compared to the cycletime of their normal counterparts, that is the cells of theadult animal from which the tumors originated, by rigorouskinetic methods. In addition to the comparison reportedabove by Reiskin and Mendelsohn (201), Dormer et cii.(57) compared cycle times of mouse epidermis and chemically induced skin carcinomas and found cycle times,respectively, of 32 and 150 hr. On the other hand, Postet at. (187, 188), comparing cycle times of rat liver cellsand chemically induced hepatoma cells, found that normalcells had a shorter cycle time, 21.5 hr, against 31 hr forhepatoma cells. Other comparisons between tumor andnormal tissues have been based on the use of daily mitoticindex or thymidine index. Bertalanify and Lau (25) foundthat daily mitotic rates of certain rat tumors were higherthan daily mitotic rates of most normal tissues, with theexception of cells of the pyloric mucosa and of the cryptsof the small intestine (23). Baserga and co-workers, onthe basis of the dilution of the label, indicated that somenormal cells proliferated at a faster rate than tumor cells,in both mice (12) and man (10), and Killmann et cii. (118),investigating in vivo the cell cycle of human leukemic cells,

found that the estimated cycle time in leukemic blast cellswas equal to or longer than the cycle time of normal neutrophil precursors. Although it may be possible that inmany instances tumor cells proliferate faster than theirnormal counterparts, the concept is gaining acceptancethat tumor cells do not necessarily proliferate faster thannormal cells (10, 56, 148, 214), and that the growth of atumor is not due exclusively to an acceleration of theproliferative process but involves other parameters ofcellular kinetics, such as the fraction of cells participatingin the proliferative pool and the amount of cell loss throughdeath or migration. This concept, expressed in 1962 byBaserga and co-workers, has found confirmation in thework of Killmann et cii. (118) who stated â€œ.. . the generalconcept of acute leukemia as a disorder of rapid prolifera




are necessary for the continuation of DNA synthesis (232),their role in the initiation of DNA synthesis is still somewhat uncertain (190, 191).

The physical state of the DNA molecule may also beimportant in temporal control of DNA synthesis. Forinstance, in vitro heat-denatured DNA, that is, singlestranded DNA (160), is often a better primer than nativeDNA (19, 134, 166), and in nondividing bacteria 90 % ofthe DNA is nonfunctional when used as primer in vitro(81). Rolfe (204) has shown that the native helicalconfiguration of DNA is altered some time prior to replication, and that part of this altered DNA is associated withproteins. It is difficult to separate this nascent DNA fromthe associated protein (22, 91). Autoradiographic studiesof Escherichia coli DNA have confirmed that the DNAmolecule, in replicating, unwinds into 2 strands (41).These findings suggest that native, resting DNA must beconverted to primer DNA before DNA biosynthesis maybegin. However, a biologic system capable of effectingthis conversion in vivo has not been identified, although ithas been suggested that such a role could be attributed tothe enzyme DNase (134, 153) or possibly another nuclease(203).

The 3rd group of factors that may be of importance ininitiation of DNA biosynthesis deals with the role of RNAand proteins, in particular with the possibility that theinitiation of DNA biosynthesis may be preceded by thesynthesis of specific RNA and proteins (191, 232). Sucha possibility is suggested by the following evidence: (a)the rate of RNA synthesis is higher in mammary glands ofmice stimulated by pregnancy than in resting mammaryglands (1), and higher in liver of weaning rodents than ofadults (226) ; (b) when liver cells begin to proliferate afterpartial hepatectomy, there is a sudden increase in the rateof RNA synthesis (38, 242). Amounts of actinomycin Dthat have no effect upon the normal rate of RNA synthesis completely suppress the rate increase that followspartial hepatectomy and produce a delay in the initiationof DNA synthesis, which usually begins 12â€”18hr afteroperation (72); (c) Lieberman et cii. (141 , 142) havedescribed changes in the pattern of RNA synthesis inrabbit kidney cortex cells cultured directly from theanimal. After explantation, these cells undergo a lagperiod of 32 hr before DNA synthesis begins. In the 1ststage of this lag period, from 0 to 12 hr, there is increasedformation of stable ribosomal RNA. In the 2nd stage,from 12 to 22 hr, there is a sharp increase in the rate offormation of nuclear RNA. This nuclear RNA hasproperties analogous to messenger RNA of bacteria, andits inhibition by actinomycin D results in complete blockbig of DNA synthesis. In the 3rd stage, from 22 to 32hr, the rate of RNA synthesis remains constant at its newelevated level, and addition of actinomycin D does notresult in inhibition of the subsequent DNA syntheticperiod. In the same system, the concentration of suchenzymes as thymidine kinase and DNA polymerase increases only at the time DNA synthesis begins; (d)Giudice and Noveffi (80) found that the rise in DNApolymerase that follows partial hepatectomy is inhibitedby actinomycin D only when the antibiotic is given 3â€”8hr after surgery, while puromycin (79), an inhibitor of

protein synthesis, is most effective 13â€”19hr after hepatectomy; (e) a similar sequence of events, leading from RNAsynthesis to synthesis of thymidine kinase to synthesis ofDNA, has been described by Hotta and Stern (103) inmicrospores of Lilium.

These findings are compatible with a model in whichinitiation of DNA biosynthesis involves DNA itself and ismediated through RNA and specific enzymes. The modelis based on generally accepted concepts that protein synthesis in cells takes place on ribosomes (77, 168, 249), andthat the sequence of amino acids, which is responsible forthe specificity of proteins, is determined by a sequence ofbases in a particlar kind of RNA molecule (151, 205). Inbacterial cells, this RNA molecule has been identified withshort-lived messenger RNA (34, 88) but in mammaliancells there is evidence that at least some of the RNAtemplates are stable (85, 95, 223). The sequence of basesin template RNA is, in turn, determined by a complementary sequence of bases in one of the DNA strands (89,93, 149, 227, 245). However, at any time, only a verysmall portion of the DNA molecule is genetically active,that is, only a small segment of the DNA strand is codingtemplate RNA for the building of specific proteins (2, 243).Thus in the adult organism most of the DNA molecule isgenetically inactive (33) and a majority of genes are repressed (65). However, repressed genes may becomeactive and code RNA templates for the coding of specificproteins (52). This situation is apparently true in enzymeinduction in bacteria. Exposure of a bacterial culture toa @-galactosidaseinducer causes an increased synthesis ofa messenger RNA that hybridizes with DNA containinga gene for @-galactosidase (96). This is interpreted asindicating that an important function of the inducer is toinactivate a repressor at the level of transcription of thecoding message from DNA to RNA. However, thepossibility has not been ruled out that regulation of enzymesynthesis may occur at the level of translation of themessage from RNA to protein (230).

As shown by Schwartz et cii. (217) and by Liebermanet cii. (72, 141, 142), inhibition of RNA synthesis a few hrbefore initiation of DNA synthesis does in fact inhibitsubsequent DNA synthesis, while inhibitors of proteinsynthesis are effective in preventing initiation of DNAsynthesis when administered immediately before replication. On this basis, the sequence of events in initiation ofDNA biosynthesis could be compared to enzyme induction in bacteria (114) and higher animals (176) or to themechanism of action of certain hormones (90), and couldbe summarized as follows: an environmental stimulus,perhaps a critical concentration of deoxynucleotides (48)or a slight modification in the physical state of the DNAmolecule, may inactivate the repressor of a genetically inactive portion of DNA. The inactivation of the repressorwould cause the synthesis of a template RNA which, inturn, would code the enzymes involved in the separation ofthe DNA strands and the polymerization of the componentdeoxynucleotides. In this context, it may be of interest toquote Swarm's conclusions (234) of a few years ago:â€œDifferentiationduring development is brought about by aprocess known as induction, the essence of which is that ashort-lived stimulus of some sort produces a relatively



Cancer Research590

long-lasting effect on the pattern of synthesis. A numberof lines of evidence suggest that stimulation of differentiated cells to divide is in fact an inductive process. . . .â€œ

CONCLUSIONS

In 1955, in a 12-page section dealing with cell division,Cowdry (49) was able to condense our knowledge of cellinterphase in a little less than 6 lines. Since then, theamount of information on metabolic activities of the interphase cell has reached such volume that a review of thiskind can only touch the most important points. Thereare still many uncertainties, but one is justified in sayingthat the life cycle of cells has been mapped out in its broadoutline, and that the details are being filled in rapidly.Our knowledge of the cell cycle has had considerableinfluence on 4 major areas of investigation: kinetics ofcellular proliferation, control of DNA biosynthesis, lifespan of cells, and cell differentiation. The last 2 areashave not been touched in the present review, but areclosely interrelated with cellular kinetics and cell division.For the student of cancer, it may be of interest to evaluatethe impact exerted on our approach to the investigation ofcancer by knowledge of the cell cycle and by permanenttagging of cells with DNA precursors. It is reasonable tosay that the kinetics of cellular proliferation, in normal aswell as in tumorous tissues, have been made more trulyquantitative and closer to the Pythagorean ideal that â€œtheprinciples of mathematics are also the principles of thingsas they areâ€•(196). At the same time, emphasis has beenshifted from the control of cell division to the control ofDNA biosynthesis, a shift that has required and will continue to require considerable reorientation in theoreticalconcepts and in experimental designs.

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Vol. 25, June 1965




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1965;25:581-595. Cancer Res Renato Baserga

A Reviewof Cell Division: The Relationship of the Cell Cycle to Tumor Growth and Control

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