CANCER RESEARCH the Relationship of the Cell Cycle To

CANCER RESEARCH

VOLUME 25 JUNE 1965 NUMBER 5

The Relationship of the Cell Cycle to Tumor Growth and Control 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-resolution autoradiography with tritium-labeled precursors of nucleic acids and proteins. After completion of mitosis, offspring of the parent cell may divide again, or may differentiate and die without further division. Dividing cells go through a series of metabolic changes which constitute the cell cycle. The cell cycle is subdivided into 4 distinct phases: (a) a postmitotic phase called G,, during which the cell synthesizes ribo nucleic acid (RNA) and proteins; (b) an S phase, during which the amount of de oxyribonuleic acid (DNA) is duplicated while protein and RNA syntheses continue; (c) a postsynthetic phase, called G2, characterized by no net synthesis of DNA and con tinued RNA and protein synthesis as in postmitotic phase; and (d) mitosis, extending from beginning of prophase to completion of telophase during which there is no DNA synthesis, protein synthesis is reduced to a minimum, and RNA synthesis is limited to early prophase and late telophase. The length of the cell cycle and its 4 phases can be obtained by appropriate experimentation with labeled thymidine, a precursor of DNA, andhigh-resolutionautoradiography. Although thelengthof the ceilcycle variesin dif ferent kinds of cells, it is shorter in certain cells of the adult animal than in some of the fastest growing tumors. Tumor growth, therefore, must involve other kinetic param eters besides speed of cell proliferation. Since these parameters are related to control of cell division and since a cell in DNA synthesis is generally a cell that has already taken the decision to divide, the various factors involved in initiation of DNA biosyn thesis are of particular interest in any attempt to explain the mechanism of tumor growth. It is possible that the initiation of DNA biosynthesis may involve DNA itself, and may be mediated through synthesis of specific RNA and enzymes.

For students of cellular proliferation, the most impor sequence of events leading from one mitotic division to the tant event in the life of a cell has always been mitosis, in next, and to understand the way in which knowledge of its classic stages from prophase to telophase (248). How these events helps to clarify some of the problems related ever, in recent years it has been found that the long inter to kinetics of cellular proliferation and tumor growth. val between 2 successive mitoses, called the interphase, included a series of metabolic events that are of importance THE CELL CYCLE in the over-all process of cell division. The orderly se Our knowledge of the cell cycle has been obtained quence of these metabolic activities, from midpoint of mostly by the use of high-resolution autoradiography mitosis to midpoint of a successive mitosis, constitutes (20, 63). The interested reader is referred to the literature what is called the cell cycle. The object of this review is for a detailed description of the technic of high-resolution to present a general outline of the cell cycle, to describe the autoradiography (14, 61, 62, 107, 124, 140, 169, 225), and of the various radioactive precursors that are most fre 1 TJSPHS Research Career Development awardee (5-K3-GM quently used to investigate the metabolic activities of cells 15, 465-05). Received for publicationSeptember2, 1964;revisedJanu (3, 4, 6, 7, 13, 14, 44, 61, 62, 64, 74, 131, 167, 194, 216, 218, ary 15, 1965. 247, 256). The most commonly used nrecursor is tritium 581 This One

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. 582 Cancer Research Vol. 25, June 1965 labeled thymidine (thymidine-3H), a deoxynucleoside by taking samples at different intervals after injection of which is incorporated exclusively into DNA (69, 200). thymidine. For 30 min or so after exposure to thymidine Since thymidine and its degradation products are soluble @Hall mitoses are unlabeled, indicating that there is a in ordinary fixatives, in which DNA is insoluble, any measurable period before mitosis during which cells do not radioactivity found in fixed tissues or cells after exposure synthesize DNA. After 30 mm, the percentage of labeled to radioactive thymidine is due to newly synthesized DNA mitoses increases and the interval between administration (5, 215), a conclusion confirmed by histochemical studies of thymidine and the time at which 50 % of mitoses are (4, 14). In addition, thymidine, when injected into intact labeled gives the duration of the premitotic phase, called animals, is either promptly incorporated into DNA or the G2 phase (104, 128) during which no DNA is synthe catabolized to nonutilizable products (189, 255). The sized. As more and more cells that were in DNA synthe time during which injected thymidine is available for sis at the time of thymidine injection enter mitosis, the incorporation into DNA is about 30 mm (119, 135), so percentage of labeled mitoses increases until 100 % of the that for all practical purposes, an in vivo exposure to mitoses are labeled (Chart 1). After a while, the per thymidine results in pulse labeling The cell cycle was centage of labeled mitoses decreases. The interval be discovered by Howard and Pelc (104). Numerous modi tween the two 50 % points on the ascending and descending fications have been introduced since their original experi limbs of the curve representing percentage of labeled ment, but the basic model remains the same. Let us sup mitoses (Chart 1) gives the duration of the phase during pose an asynchronous population of cells, such as the which DNA is synthesized, called the S phase (104, 128, lining epithelium in the crypts of the small intestine of 197, 198). The length of the entire cell cycle can be mice. When thymidine.-H' is injected into mice, cells estimated on the basis of the time elapsing between 2 suc that are synthesizing DNA at the time of injection take up cessive peaks of labeled mitoses (70, 219). In Chart 1, the thymidine and become labeled. An autoradiograph of a interval between the midpoints of the 1st and 2nd peaks is section of small intestine, 30 mm after injection of thy 12 hr and the length of the cell cycle is then estimated at midine, discloses 3 types of epithelial cells in the crypts: 12 hr. The duration of G2 is 1 hr, and the length of S phase, cells in mitosis (all unlabeled), unlabeled interphase cells, 7 hr. Since mitoses usually last less than 1 hr (71, 99, and labeled interphase cells. The 1st bit of information we have obtained is that DNA is synthesized during inter 139, 250) another phase must be postulated to complete the phase and not during mitosis, since all cells in mitosis are 12 hr of the cell cycle. The existence of this phase is also unlabeled. But because of the short period of time during indicated by the decrease in percentage of labeled mitoses which thymidine is available, and the fact that DNA is after the 8th hr, which suggests a period, preceding DNA stable (21, 97, 116, 150, 202), we can now follow the fate of synthesis, during which no DNA is synthesized. The cells that were in DNA synthesis at the time of injection duration of this period, called the G1 phase, can be ob

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8 12 TIME IN HOURS AFTER INJECTION OF TRITIATED THYMIDINE CHART 1.â€”Percentage of labeled mitoses in the lining epithelium of the crypts of the small intestine of mice at various intervals after a single injec tion of â€˜H-thymidine (see explanation in the text). Reproduced, with permis sion, from the paper by Lesher et at. (137). The mice were injected at 0 time with 50 pc of thymidine-'H. In this particular experiment, the cell cycle was studied in the intestine of chronically irradiated animals. One of these groups was chosen for the present illustration, because the amount of varia tion at the various points was remarkably low. However, the curve for nor ma! mice was essentially similar.

of the label, increases for a time equal to T8 and then reaches a plateau. A variant of this approach, described by Stanners and Till in tissue cultures (228), has been applied in vivo by Koburg (123). T, can also be calculated by exposing cells to 2 different pulses, one of thymidine â€˜4Cand the other of thymidine-3H (16, 186, 252). Cells labeled with thymidine-'4C only can be distinguished from MI'FO$I@ cells labeled with thymidine-@H only or with both isotopes CELL$ by 2-emulsion autoradiography (17), and the length of the S phase can be calculated from appropriate equations (252). Although each of these methods is useful in particular situations, the one described in Chart 1 must NON -DIVIDING still be considered the most accurate and the method of CuLL choice whenever feasible (197). The length of the S phase in mammalian cells is often

CHART 2.â€”The life cycle of cells. At completion of mitosis, very close to 8 hr (42, 159, 175, 228, 254). However, the daughter cells may go through another life cycle (G1, S and G2), number of exceptions to this rule has been rapidly increas or become nondividing cells, destined to die without any further ing in the past few years. Certain near-tetraploid ascites division. tumors of mouse have a T8 of 11â€”12hr (8, 55, 102), and other instances of T8 of unusual length have been described tained by subtracting from the entire length of the cell in cells of germinal centers of rat spleen, 4.5 hr (66); in dog cycle the duration of G2 + S + mitosis. myeloblasts, 5 hr (179) ; in epithelial cells of human colon, The cell cycle (104, 128, 197) may then be defined as the 9 hr (145) ; in transplantable mammary tumors of mouse, interval between midpoint of mitosis in the parent cell 10 hr (159); in epithelial cells of mouse forestomach, 13.5 and midpoint of the subsequent mitosis in the daughter hr (253); in mouse spermatogonia, 14 hr (163) ; in alveolar cell (Chart 2). If one of the sister cells is destined not to cells of mouse mammary gland, 20 hr (35); and in epithe divide again, it leaves the cell cycle and becomes a non hal cells of mouse ear epidermis, 30 hr (220). T@may even dividing cell that may eventually die or persist for the vary in the same tissue under different conditions, as in entire life of the organism without dividing (Chart 2). the intestinal epithelium of mice, where T@in chronically The lining epithelium of the small intestine can be used to irradiated mice is 1 hr shorter than in controls (137). In illustrate an admixture of dividing and nondividing cells, Ehrlich ascites cells T8 is shorter in diploid than in near although similar admixtures occur in other tissues (181). tetraploid lines (55). Actually, in the same hypotetra DNA synthesis and cell division occur only in the crypts ploid line, both T8 and T@may vary with the sex of the (136, 198). From the crypts, cells migrate to the villus, recipient mouse. In the Ehrlich tumor described by where they become nondividing cells, destined to be shed Baserga and co-workers, T8 and T@last, respectively, 11 into the intestinal lumen without any further mitotic and 18 hr in male mice (8) and 17 and 36 hr in females (9). division. Thus, the original assumption of the constancy of the S THE 4 PHASES OF THE CELL CYCLE2 phase in mammalian cells is no longer tenable. DNA synthesis is only one of several synthetic activities S phase.â€”Wehave seen that DNA synthesis takes place that take place during the S phase. Cells synthesize in a discrete period of interphase, called the S phase (104, RNA throughout the entire interphase, as evidenced by 236, 246). There are several ways of determining the the fact that nearly all interphase cells are labeled by a average length of the S phase, T.. The most commonly brief exposure to a radioactive RNA precursor (94, 237). used is the one described above in Chart 1 (70). Another In the ciliated protozoan Euplotes, Prescott has shown that way of determining T8 is by continous labeling (251). If DNA and RNA syntheses are mutually exclusive, as cells are constantly exposed to tritiated thymidine, as in evidenced by the lack of uptake of RNA precursors in infusion technics (180) or in tissue cultures (228), the regions of nuclei at the time of DNA replication (193, 195). grain count per cell, that is, the autoradiographic intensity It is also known that RNA polymerase is more active when

2 We have adopted the nomenclature and definitions that were primed by native nonreplicating DNA than by single agreed upon among the participants to the Guinness Symposium stranded DNA (73, 111). However, in mammalian cells, on Cell Proliferation (197). Besides the definitions given above of because of asynchrony in DNA replication (143), the rate the cell cycle and its phases or compartments (5, G1, G,, M), of uptake of RNA precursors is substantially the same in these include the following conventions : N@means the number of cells in a compartment, and therefore N8 is the number of cells cells in DNA synthesis and in other cells in interphase (6). in the S phase, N@ the total number of cells in the population, The rate of protein synthesis, as measured by uptake of etc. Similarly, T@is the transit time or the time elapsed between labeled amino acids, is increased during the S phase. This entering and leaving a compartment : e.g., T@is the length of cell was shown directly in protozoa by Prescott (192), in cycle, T. the length of the S phase, etc. The thymidine index, I, is defined here as the percentage of cells labeled within a onion root meristem by Olszewska (173), and in mam 30-mm interval after a single injection of radioactive thymi malian cells by Baserga (7) and Baserga and Kisieleski dine(13).In theabove notation: (13). This conclusion is also supported by the findings of lOON, Bloch and Godman (29) and Dc (54) on histone synthesis Iâ€” N@ (A) and by those of Lipkin et al. (144) that the uptake of radio

active leucine by the small intestine is higher in crypt cells synthesized DNA without proceeding to mitosis, and a than in villus cells, where no DNA synthesis occurs. similar situation has been reported by Owen and Mac However, DNA and protein synthesis may proceed Pherson (174) in rabbit osteocytes. These cells arrested independently from each other, as shown by Maaloe (147) in the G2 phase must contain at least twice the amount of in bacteria, and by Rueckert and Mueller (207) and DNA of interphase diploid cells, that is, they are polyploid Rusconi (208) in mammalian cells. cells, a condition that is frequently found in liver cells and Postsynthetic phase.â€”In this phase, the G2 phase, cells other tissues of rodents (39, 105, 235, 236) and is the rule synthesize RNA and proteins but not DNA (6, 7, 94, 170, in myocardial cells of man (209). Despite these excep 192, 237). The length of the postsynthetic phase is fairly tions, in most instances, cells rest in the G1 period of the @ constant in mammalian cells, ranging from to 1@hr (67, cycle (165). 175, 197, 228). There are exceptions, like cells of the ear Cellular sites of nucleic acid and protein syntheses.â€”There epidermis of mouse (220), pre-enamel cells of the develop is general agreement that DNA is synthesized in the ing enamel organ in teeth of young rabbits, in which the nucleus, with the exception of DNA viruses, which can G2 phase lasts from 6 to 9 hr (229), some near-tetraploid replicate in the cytoplasm (112), and possibly of the DNA ascites tumors of mouse with a G2 phase of about 6 hr (8, fraction located in mitochondria (76). It is also agreed 55), and mouse spermatogonia, that have a variable G2 that proteins are synthesized in the nucleus as well as in time (163). the cytoplasm (14, 172, 247), but there is still some un Mitosis.â€”The duration of mitosis has been estimated to certainty as to the sites of synthesis of RNA. This subject vary in different tissues from 30 mm to 2.5 hr (71, 99, 139, has been reviewed recently by Graham and Rake (84). 201, 250). It may be mentioned briefly that, during Briefly, we can say that the bulk of evidence indicates that mitosis, protein synthesis reaches a minimum (7, 13) and RNA is synthesized in the nucleus, from which it migrates RNA synthesis is limited to early prophase and late to the cytoplasm (83, 223). A large portion of RNA has a telophase (51, 194). The end result of mitosis is reduction high turnover rate and breaks down rapidly after being of the amount of DNA per cell to the diploid value (236) synthesized (95). Soluble RNA and ribosomal RNA are characteristic of the species (30). also synthesized in the nucleus (40, 45, 78), ribosomal Postmitotic phase.â€”At completion of mitosis, the cell RNA actually in the nucleolus (36, 154, 183, 184), and enters the G, phase. Of the 4 phases of the cell cycle, this then transferred, at least in part, to the cytoplasm (223). is the most variable in length (67, 159, 238). It is very short, practically undetectable by ordinary methods, in the THE STEADY STATE SYSTEM slime mold Physarum polycephalum (170), in the sea urchin Many tissues of the adult animal are in a steady state in embryo (153), in Ehrlich ascites cells growing in the respect to the total number of cells. This means that not peritoneal cavity of male mice (8), and in pre-enamel cells only does the total number of cells remain constant, but of young rabbits (229), while in other mammalian cells it the number of cells in each compartment also remains may last from a few hours to several months (181). It is constant. A good example of a steady state system is the also the phase in which most cells with a long cycle time lining epithelium of the mucosa of the small intestine (132, stop for an indefinite time as evidenced by the following 198). The epithelial cells that line the intestinal mucosa findings : (a) in tissue cultures the S and G2 phases are belong to 2 distinct compartments : the crypt and the constant and variations in length of the cell cycle depend villus. The crypt is where cell division occurs (28) and it upon variations in length of the G1 period (224, 238); (b) is called the progenitor compartment. The villus is the in adrenal gland of rats, the length of the cell cycle varies functional compartment, where cells do not divide. While from 80 hr in cells of the glomerulosa to about 250 hr in cells from the crypt migrate to the vifius, cells almost those of the outer fasciculata. Since the length of 5, G2, never return from the functional into the progenitor and mitosis appears to be the same, the phase that varies compartment (198). Each time a cell in the crypt divides in length must be the G1 (67); (c) in regenerating liver, the into 2 daughter cells, another cell migrates from the crypt curve for percentage of hepatocytes in DNA synthesis at into the villus. Similarly, in the villus, for every cell different intervals after partial hepatectomy precedes by entering it from the crypt, another cell leaves at the tip about 6â€”8hr, both in initial elevation and peak incidence, and is shed into the intestinal lumen, where it dies (132, the curve for mitotic index (87, 138). There are, however, 136, 198). If the number of cells remains constant, it exceptions. Gelfant (75) was the first to suggest that some follows that 1 of the 2 cells produced by cell division is cells of mouse ear epidermis may be arrested, not in G1,but destined to die without dividing again. It would be in the G2 phase, from which they entered mitosis after a tempting to speculate that, of 2 sister cells produced by suitable stimulation. Cameron and Cleffmann, taking each division, 1 divides again and the other migrates to the advantage of the fact that cell proliferation, depressed by villus and dies. This is not necessarily true and it is starvation, can be stimulated by refeeding (133, 164), have possible that, in some instances, both sister cells divide determined variations in mitotic and thymidine indices of again, while in other instances both sister cells leave the starved chickens with time after refeeding. These authors crypt without any further division. Leblond (130) were able to show that practically all epithelial cells of the inclines to the opinion that, at least in esophagus and duodenum were resting in G1, while a sizable portion of intestinal epithelium, the decision as to whether a pro esophageal cells were in G2, from which they proceeded liferating cell will differentiate or continue to divide takes directly to mitosis (43). Starkey (229) also found that, in place during interphase and depends on local environment. rabbit teeth, enamel is elaborated by cells that have The various parameters of cell division and migration in

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. BASERGAâ€”Ce11 Cycle and Tumor Growth 585 the lining epitheium of the intestine have been described mining T@,using both thymidine-3H and colchicine, has in detail, in mouse by Quastler and Sherman (198) and been introduced recently by Van't Hof and Ying (244) Lesher et al. (136), in rat by Leblond and Stevens (132), and applied to root meristems of Pisum satwum. Among and in man by Lipkin et al. (145), and are substantially the methods using only high-resolution autoradiography similar in various species. Analogous systems are found and labeled DNA precursors, the one illustrated in Chart elsewhere in the body, for instance in squamous epithelium 1 probably represents the most straightforward way of lining the skin and mucosae of internal cavities (182). determining T@. Unfortunately, it is not always feasible Steady state systems like bone (113) and bone marrow (32, because, as mentioned above, some cell populations 177, 180) are rather more complicated, but they are desynchronize in 1 generation and the 2nd wave of labeled also characterized by the fact that the total number of mitoses fails to materialize (108). The possibility exists cells remains constant, with cell production balanced by that failure of the 2nd wave of labeled mitoses to materi cell loss. In the rat, several cell populations, like those of alize may be due to a statistical error inherent in a small the kidney, pancreas, or skeletal muscle, that in man are number of observations. For instance, in his original considered in a steady state system because the weights description of 28 mammary tumors of C3H mice, Mendel of the organs plateau in adult life (199), are in fact slowly sohn (155) could not detect a 2nd wave of labeled mitoses, expanding until 1 year of age (60). During this time, the but when an additional 92 tumors were investigated, he only tissue that shows no increase in cell number is cere was able to recognize that the 1st wave of labeled mitoses bellum (60). After 1 year of age, however, these expand was followed by 2 other recognizable and equally spaced lug cell populations also become steady state systems (130). waves (158). It is therefore conceivable that the method described by Fry â‚¬1al.(70) may turn out to have a wider LENGTH OF CELL CYCLE application than presently allowed. We have defined T., as the mean transit time around the Another way of determining T@is based on the fact that entire cell cycle, that is, the time elapsed between the mid T, can be determined independently in an accurate way points of 2 successive mitoses. Sisken and Kinosita (224) (see above), and that the relation between T, and T@can have determined the length of the cell cycle of individual be expressed by the equation: cells in tissue culture by time-lapse microcinematography. These authors followed individual cells in a colony of Na/Nc = T,/TC (B) human amnion cells from one anaphase to the next and where N, is the number of cells labeled by a pulse-exposure found T@= 21.6 hr Â± 0.5. In vivo measurements of to thymidine-3H and N@is the total number of cells in the individual cells are not available yet, and thus, T@usually population. This equation is valid in an ideal system gives the mean transit time for a cell population without (197), but, in practice, it can be used in only a few selected any knowledge of individual variations. Variations in instances. Apart from the fact that N, may undergo length of the cell cycle by individual cells may make T@ diurnal fluctuations (68, 185), a serious shortcoming of rather meaningless. For instance, if a population of cells Equation B is that it requires the assumption that all cells consists of 2 distinct sub-populations, one with a long cell in the population participate in the proliferative process. cycle and a second one with a short cell cycle, the mean Actually, most proliferative compartments contain ad cycle time T@may mean very little. Such populations mixtures of cells which are not going to reproduce. The have been described in the forestomach of mice by Wolfs ratio NI/NC will then be less than the ratio NI/NP, where berg (253) and in macrophages of pulmonary alveoli of N@ is the total number of cells in the proliferative cycle mice by Shorter et al. (222). Even in a more homogeneous (157). The result is an overestimate of the length of the population, individual cycle times of cells may vary cell cycle. For instance, T, in the epitheial cells lining greatly about the mean although the direct measurements the small intestine of mice is 7 hr. The ratio N,/N@ is of Sisken and Kinosita (224) indicate small variations, a about 0.065 if determined on all epithelial cells lining the possibility confirmed by the findings that the length of T@ intestinal epithelium, that is, epithelial cells of the crypts may be measured by the interval between 2 successive as well as nondividing cells of the vffli. If only crypt peaks of labeled mitoses (70), and that in bacteria the epithelial cells are counted, the ratio is 0.45. By rear cycle time has a normal distribution (126). However, in ranging and substituting in Equation B, one would obtain mammary tumors of mice, the 2nd and 3rd waves of 2 values of Tc of 107 and 15 hr, respectively, a substantial labeled mitoses are lower than the 1st peak, and the cells difference. When using Equation B it is therefore have completely desynchronized by the 4th generation. important to know N,,, defined by Mendelsohn (155, 157) Together with the damped 2nd wave of labeled mitoses as the Growth Fraction, or the fraction of cells in the total described by Johnson in a transplantable mouse sarcoma population that participate in the proliferative process. (108), they indicate a considerable amount of variance in Since N, = pNc (where p is the fraction of proliferating cycle times of individual cells, at least in some populations cells), the problem is how to determine p in a cell popula of cells. However, until methods become available for tion in vivo. One way around it is to obtain 7.'@from the determining T, of individual cells in vivo, one should not equation: reject as meaningless the measurements that can be ob tamed by the methods to be described below. (C) The classicmethodsforstudyingsomeofthe parameters Tc M*/M of cell proliferation have been based on the use of col where M* is the number of labeled mitoses and M the chicine (24, 129, 231), and a combined method for deter total number of mitoses (157). The equation requires an

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. 586 Cancer Research Vol. 25, June 1965 independent determination of T,, followed by a deter restrict its use to selected situations. In practice it is mination of M*/M after the labeled cells have desyn often difficult to differentiate nonproliferating cells from chronized. A difficulty with this method is that if one cells with a very long cycle, the G0 cells of Patt and waits too long for cells to become properly desynchronized, Quastler (181). For instance, there are cells with a long the concomitant dilution of the label makes recognition of cell cycle which are still capable of DNA synthesis and labeled cells increasingly uncertain. On the other hand, which, under proper conditions may shorten the length of if cells desynchronize rapidly, the T@obtained will rep the cycle. A remarkable example of this kind is the liver, resent a mean to which individual cells confirm very ordinarily a rather quiescent tissue with a low thymidine poorly (157). An alternative is to determine the fraction index (11, 58, 162, 221), which can markedly increase its of proliferating cells, p, from the number of cells that are capacity for DNA synthesis during the regenerative phase labeled by continuous exposure to thymidine-'H provided that follows partial hepatectomy (37, 38, 87, 138). Epi only the progenitor compartment is taken into considera thelial cells of the anterior surface of the crystalline lens tion. In tissue cultures, p can be determined by incubat (92), basal cells of the epidermis (98), smooth muscle cells ing cells with thymidine-3H and observing the number of of arteries and arterioles (50), regenerating skeletal muscle cells that become labeled over an extended period of time cells (27, 120), and kidney cells (100) are also cells known (228). An in vivo modification was applied by Baserga to respond to an appropriate stimulus with an increase in et al. (15) to Ehrlich ascites cells growing in lungs of mice. the number of cells synthesizing DNA and a shortening of Tumor-bearing animals were injected every 4 hr with the cycle time. Other cells, like keratinizing epithelial thymidine-3H, and since all cells were found to be labeled cells of the upper layer of the epidermis, various neurones, after 6 injections, it was concluded that all cells partici cells of the nervous layer of the retina, and polymorpho pated in the proliferative process, that is p = 1. Con nuclear leukocytes are not known to be capable of taking tinous incubation or repeated injections can be replaced up thymidine, at least in the adult mouse (58, 162, 221), by continous infusion of thymidine-3H (156). When the and should be considered as cells that have left the cycle. latter method was used in mammary tumors of C3H Because of the limitations mentioned above, most of the mice, a number of morphologically intact tumor cells re measurements of T@reported in the literature must be mained unlabeled even after infusions as long as 10 days. considered with caution. A large majority of these This indicated that p in these tumors was considerably measurements are simply based on determinations of I below 1. Precise measurements showed p = 0.18 (158). and T, and the use of equation B, with its attendant in Another method for determining Tc is based on the conveniences. In only a few instances has T@actually dilution of the label. In a population of labeled cells in been determined, either by the method of Fry et al. (70) which all cells divide, the specific activity of DNA de or by using N, to modify equation B. Some of these creases in direct proportion to the number of cell divisions measurements and estimates in man and mice are sum (150, 202). In autoradiographs, the decrease in specific marized in Table 1. The figures given for human tissues activity of DNA can be followed by determining the mean are all approximate estimates but in mice we have some grain count of cells pulse-labeled with thymidine-'H. measurements that can be accepted with greater confi Using this principle, Baserga et at. (18) have proposed a dence. For instance, the length of the cell cycle in the method based on the distribution of grain counts that epithelium of the small intestine has been determined by gives at the same time p and T@. The method requires 2 the method of Fry et at. (70), and that of the Ehrlich determinations of the mean and distribution of the grain ascites tumor by using Equation B, since, in this tumor in counts of a cell population at different intervals after a the exponential phase, p = 1 (146). It will be noted that single injection of thymidine-3H. The 1st determination the length of the cell cycle is shorter in the non-growing must be taken after the labeled cells have undergone the normal adult tissue than in the rapidly growing Ehrlich 1st division following exposure to thymidine. From the tumor. grain count mean and variance at time t0. and at time to + @It, it is possible to obtain the proportion of cells KINETICS OF TUMOR GROWTH that have not divided, a, that have divided once, b, and The simplest equation that describes the growth of a that have divided twice, c, in the interval L@t(18). tumor is of the exponential type y = bx, and it seems to The length of the cell cycle, T@, of the proliferating fit best some ascites tumors. Patt and Blackford (178) subpopulation is then given by: used it to describe the 1st week of growth of Krebs ascites I@t tumor, Maruyama (150) fitted it to the growth of LSA T,=@â€”@- (D) ascites tumor, and Baserga (9) found an exponential pattern of growth in Ehrlich ascites tumor growing in the The method requires that the interval @tbe > T@< 2T@. peritoneal cavity of mice, regardless of the method used to It gives a good first approximation of both p (in this case, estimate growth, e.g., number of tumor cells, dry weight of 1 â€”a) and T@but it again requires a rather rapid desyn tumor cells, or total amount of tumor DNA. In all these chronization of labeled cells. A more elaborate statistical cases, however, the curves flatten after the 1st week or so method has been proposed recently by Tyler and Baserga of growth, usually when the total number of tumor cells (241), but it would go beyond the purpose of the present per mouse reaches a figure of 500â€”600million cells (9, 178). discussion. Probably for this reason, Klein and Revesz (121) thought In conclusion, while there are several methods that can that the growth curve of Ehrlich ascites carcinoma was be used to determine T@,each of them has limitations that better expressed in terms of the cube roots of tumor cell

TABLE 1 dN ln 2 (F) ESTIMATED LENGTH OF THE CELL CYCLE IN DIFFERENT dt Tc TYPES OF CELLS IN Vivo Since both T@and N can be determined experimentally, TissueEstimatedlengthReferenceMAN:Epidermis1.5 dN/dt can be obtained by substitution. If T@is unknown, dN/dt can be calculated from the equation given by Stanners and Till (228). mo110Polychromatophil These manipulations are valid in an exponentially hr179Epithelium normoblasts15â€”18 growing population, but since only ascites tumors in their hr145Epitheliumof large intestine25 initial stages grow exponentially, exponential equations are hr46Carcinomaof large intestine53 of limited value in describing the actual growth of a solid hr10Leukemicof colon, metastases27 tumor (157). Mendelsohn (158) has reiterated that solid hr118Carcinomablast cells50â€”80 tumors, like mammary tumors of mice and human tumors, hr10Carcinomaof stomach66 do not conform to an exponential pattern of growth, and hr10Multiple of colon, metastasis75 that different equations must be used to describe the days117Carcinomamyeloma cells2â€”6 growth pattern of tumors in which the nonproliferating days10Glioblastomaof liver10 mo109Ovarian multiforme1 subpopulation of tumor cells constitutes a substantial mo110Ovariancystadenoma1.5 part of the total cell population. A plausible model for mo13Carcinomacarcinoma1.5 such a mixed population is the stochastic model of cell mo110Leiomyomaof breast3 proliferation and differentiation proposed by Till et al. mo110MOUSE:Epitheliumof spermatic cord5.5 (239), based on a â€œbirth-and-deathâ€• process, for details of which we must refer the reader to the original paper. The hr136Epitheliumof small bowel11.6 situation is further complicated by the fact that a tumor hr57Bronchialof epidermis150 is not a closed system in which there is no cell loss from the days122Trophoblastepithelium18 compartment. To begin with, necrosis, that is, cell death, hr42centaEhrlichcells, 12th-day pla 15 is a frequent occurrence, especially in solid tumors. In hr8Skin ascites tumor18 addition to cell loss because of necrosis, of individual cells hr57Melanomacancer32 or of entire portions of the tumor, cell loss may occur be days26Breast (transplanted)2.5 cause of migration of cells out of the main proliferative cancer1â€”3.5 days159 compartment. Metastases are instances of cell migration, although the number of metastasizing cells is usually negligible (47, 257) and secondary colonies established by numbers. If a generalization must be drawn from the tumor cell emboli can be considered as independent growth curves proposed for ascites tumors, it is perhaps compartments. More important is migration of cells in the conclusion reached by Patt and Blackford (178) that surrounding tissues, which is of such magnitude in some the initial growth pattern may be approximated by an ascites tumors as to produce an actual decrease in total exponential fit and a portion of the curve by a cube root number of cells in late stages of growth, although some cell transform, and that neither the exponential nor the cube proliferation is still taking place (9, 59). root transformation accounts for the entire growth pat The growth pattern of a tumor is important not only in tern. any attempt to understand how a tumor grows, but also However, even if we accept an exponential equation to because the kinetics of the system require corrections in describe the initial pattern of growth of some ascites the equations that we have found useful for measuring T@ tumors, a cube root equation seems to apply best to the in a steady state system. For the purpose of the present growth of solid tumors (152, 213, 214), especially those in discussion, we shall limit ourselves to corrections necessary which, because of extensive central necrosis, growth in case of exponential growth. Although this is not always depends mainly on the peripheral cellular layers of the correct, as mentioned above, we should remember that the tumor nodule. Actually, Mayneord (152) believed that error may be of little value in comparative studies. For the cubic root pattern of growth transforms into an ex instance, Reiskin and Mendelsohn (201), using an equa ponential one when necrosis is reduced to a minimum and tion valid for linear growth, estimated the cell cycle time all cells participate in the proliferating process. Both of induced epithelial tumors of the hamster pouch at 20.7 equations have been summarized by Mendelsohn (157) in hr, and assuming an exponential growth at 17.5 hr. The a single differential equation: difference may seem substantial but it becomes much less impressive when it is compared to the cell cycle time of the

@ = (E) normal counterpart,the squamousepitheliumof the hamster pouch, estimated at 142 hr. Several corrections have been proposed in the case of where N is the number of cells, t is time, and K a growth exponentially growing populations (59, 108, 135), most of constant. The expression dN/dt is the growth rate of a which are useful in particular situations. We have found cell population at any instant. In the case of an ex convenient, in studying the Ehrlich ascites tumor, to ponentially growing population, b in Equation E is equal to 1, and K = X = in 2/T, where T is the doubling time adopt the correction proposed by Johnson (108), the (108). Equation E may now be written: general statement of which is expressed in the equation:

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. 588 Cancer Research Vol. 25, June 1965 convenient, in studying the Ehrlich ascites tumor, adoption tion . . . should be discarded as a general concept of the of the correction proposed by Johnson (108), the general nature of leukemia,â€•and, more recently, in the findings of statement of which is the equation: Post and Hoffman (187, 188). The picture may be more difficult to comprehend than the traditional uncomplicated (i_t@) T I@=ln2â€¢2 T@ .@ (G) picture of tumor cells proliferating at full speed, but it is probably closer to the truth and opens new horizons for a where I@is the fraction of the total population in a given proper design of chemotherapeutic or radiotherapeutic phase, T1 the phase duration, and t the average age of cells procedures. in that phase, expressed in terms of T@. For instance, to determine the fraction of the cell cycle that the Ehrlich CONTROL OF DNA BIOSYNTHESIS ascites cells spend in S phase, the equation becomes: An increased production of cells, caused by either shortened length of the cell cycle or increased size of the I = in 2 . 2@''@ . (H) proliferating pool, ultimately means an alteration in the control of cell division. In previous sections, we have seen where I is the thymidine index and T, the S-transit time that a cell that has synthesized DNA is generally a cell (9, 108). The equation can be solved for T,/T@to obtain that has already made the decision to divide. Control of the fraction of the cell cycle that the Ehrlich cells spend in cell division (233, 234) then becomes control of the initia DNA synthesis. If the duration of the S phase is known tion of DNA biosynthesis. Factors that may intervene in (see above), T@can be obtained, and conversely, T, can be temporal regulation of DNA biosynthesis (127) are estimated if T@is known. conveniently grouped in : (a) factors that may control DNA Comparison between tumors and normal tissves.â€”As can synthesis through the availability of DNA precursors and be seen from Table 1, the cell cycle time of the fastest related enzymes; (b) the physical state of the DNA growing experimental tumors is actually longer than the molecule; and (c) regulatory mechanisms related to the cycle time of some normal tissues. In only 3 cases has synthesis of RNA and proteins. the cycle time of tumor cells been compared to the cycle DNA synthesis in vitro requires primer DNA, the 4- time of their normal counterparts, that is the cells of the component deoxynucleotides, and the enzyme, DNA adult animal from which the tumors originated, by rigorous polymerase (125), and it is reasonable to assume that the kinetic methods. In addition to the comparison reported availability of precursors and enzyme is a necessary condi above by Reiskin and Mendelsohn (201), Dormer et cii. tion for the synthesis of new DNA. However, it may well (57) compared cycle times of mouse epidermis and chem be that both precursors and enzymes are manufactured in ically induced skin carcinomas and found cycle times, response to a message issued by the cell after it has taken respectively, of 32 and 150 hr. On the other hand, Post the decision to synthesize DNA. Several kinds of et at. (187, 188), comparing cycle times of rat liver cells evidence suggests that the concentration of DNA pre and chemically induced hepatoma cells, found that normal cursors and related enzymes may be determinant in the cells had a shorter cycle time, 21.5 hr, against 31 hr for initiation of DNA synthesis: (a) deoxynucleotides, a! hepatoma cells. Other comparisons between tumor and though present in small amounts in tissues of the adult normal tissues have been based on the use of daily mitotic animal (210), are found in increased amounts in proliferat index or thymidine index. Bertalanify and Lau (25) found ing tissues, like regenerating liver (212) and tumor tissue that daily mitotic rates of certain rat tumors were higher (206, 211); (l@) pyrimidines, purines, and nucleotides than daily mitotic rates of most normal tissues, with the decrease the helix-coil transition temperature of thymus exception of cells of the pyloric mucosa and of the crypts DNA, that is, they favor the transition of the DNA of the small intestine (23). Baserga and co-workers, on molecule from the 2-stranded helical form to the single the basis of the dilution of the label, indicated that some stranded coiled form (240); (c) the concentration of normal cells proliferated at a faster rate than tumor cells, enzymes concerned with the phosphorylation of thymidine in both mice (12) and man (10), and Killmann et cii. (118), and the polymerization of deoxynucleotides, such as investigating in vivo the cell cycle of human leukemic cells, thymidine kinase, thymidylate synthetase, and DNA found that the estimated cycle time in leukemic blast cells polymerase, increases in proliferating tissues (53, 86, 106, was equal to or longer than the cycle time of normal neutro 171). However, in regenerating liver, the activity of these phil precursors. Although it may be possible that in enzymes reaches a maximum after the rate of DNA syn many instances tumor cells proliferate faster than their thesis has begun to decline (31, 101). Similarly, in normal counterparts, the concept is gaining acceptance cultures of L cells, DNA polymerase activity increases that tumor cells do not necessarily proliferate faster than when DNA synthesis is inhibited and decreases when normal cells (10, 56, 148, 214), and that the growth of a DNA synthesis is allowed to proceed (82). An interesting tumor is not due exclusively to an acceleration of the alternative has been suggested by Kielley (115), who found proliferative process but involves other parameters of that thymidylate kinase activity was about equal in cellular kinetics, such as the fraction of cells participating normal mouse liver and in hepatomas, but in the former in the proliferative pool and the amount of cell loss through the activity was particle-bound and did not become evi death or migration. This concept, expressed in 1962 by dent until after disruption of the cellular constituents. If Baserga and co-workers, has found confirmation in the one had to evaluate the present evidence on the concentra work of Killmann et cii. (118) who stated â€œ... the general tion of enzymes and precursors in relation to DNA syn concept of acute leukemia as a disorder of rapid prolifera thesis, one would be tempted to conclude that while these

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. BASERGAâ€”Cell Cycle and Tumor Growth 589 are necessary for the continuation of DNA synthesis (232), protein synthesis, is most effective 13â€”19hrafter hepatec their role in the initiation of DNA synthesis is still some tomy; (e) a similar sequence of events, leading from RNA what uncertain (190, 191). synthesis to synthesis of thymidine kinase to synthesis of The physical state of the DNA molecule may also be DNA, has been described by Hotta and Stern (103) in important in temporal control of DNA synthesis. For microspores of Lilium. instance, in vitro heat-denatured DNA, that is, single These findings are compatible with a model in which stranded DNA (160), is often a better primer than native initiation of DNA biosynthesis involves DNA itself and is DNA (19, 134, 166), and in nondividing bacteria 90 % of mediated through RNA and specific enzymes. The model the DNA is nonfunctional when used as primer in vitro is based on generally accepted concepts that protein syn (81). Rolfe (204) has shown that the native helical thesis in cells takes place on ribosomes (77, 168, 249), and configuration of DNA is altered some time prior to replica that the sequence of amino acids, which is responsible for tion, and that part of this altered DNA is associated with the specificity of proteins, is determined by a sequence of proteins. It is difficult to separate this nascent DNA from bases in a particlar kind of RNA molecule (151, 205). In the associated protein (22, 91). Autoradiographic studies bacterial cells, this RNA molecule has been identified with of Escherichia coli DNA have confirmed that the DNA short-lived messenger RNA (34, 88) but in mammalian molecule, in replicating, unwinds into 2 strands (41). cells there is evidence that at least some of the RNA These findings suggest that native, resting DNA must be templates are stable (85, 95, 223). The sequence of bases converted to primer DNA before DNA biosynthesis may in template RNA is, in turn, determined by a comple begin. However, a biologic system capable of effecting mentary sequence of bases in one of the DNA strands (89, this conversion in vivo has not been identified, although it 93, 149, 227, 245). However, at any time, only a very has been suggested that such a role could be attributed to small portion of the DNA molecule is genetically active, the enzyme DNase (134, 153) or possibly another nuclease that is, only a small segment of the DNA strand is coding (203). template RNA for the building of specific proteins (2, 243). The 3rd group of factors that may be of importance in Thus in the adult organism most of the DNA molecule is initiation of DNA biosynthesis deals with the role of RNA genetically inactive (33) and a majority of genes are re and proteins, in particular with the possibility that the pressed (65). However, repressed genes may become initiation of DNA biosynthesis may be preceded by the active and code RNA templates for the coding of specific synthesis of specific RNA and proteins (191, 232). Such proteins (52). This situation is apparently true in enzyme a possibility is suggested by the following evidence: (a) induction in bacteria. Exposure of a bacterial culture to the rate of RNA synthesis is higher in mammary glands of a @-galactosidaseinducer causes an increased synthesis of mice stimulated by pregnancy than in resting mammary a messenger RNA that hybridizes with DNA containing glands (1), and higher in liver of weaning rodents than of a gene for @-galactosidase (96). This is interpreted as adults (226) ; (b) when liver cells begin to proliferate after indicating that an important function of the inducer is to partial hepatectomy, there is a sudden increase in the rate inactivate a repressor at the level of transcription of the of RNA synthesis (38, 242). Amounts of actinomycin D coding message from DNA to RNA. However, the that have no effect upon the normal rate of RNA synthe possibility has not been ruled out that regulation of enzyme sis completely suppress the rate increase that follows synthesis may occur at the level of translation of the partial hepatectomy and produce a delay in the initiation message from RNA to protein (230). of DNA synthesis, which usually begins 12â€”18hr after As shown by Schwartz et cii. (217) and by Lieberman operation (72); (c) Lieberman et cii. (141 , 142) have et cii. (72, 141, 142), inhibition of RNA synthesis a few hr described changes in the pattern of RNA synthesis in before initiation of DNA synthesis does in fact inhibit rabbit kidney cortex cells cultured directly from the subsequent DNA synthesis, while inhibitors of protein animal. After explantation, these cells undergo a lag synthesis are effective in preventing initiation of DNA period of 32 hr before DNA synthesis begins. In the 1st synthesis when administered immediately before replica stage of this lag period, from 0 to 12 hr, there is increased tion. On this basis, the sequence of events in initiation of formation of stable ribosomal RNA. In the 2nd stage, DNA biosynthesis could be compared to enzyme induc from 12 to 22 hr, there is a sharp increase in the rate of tion in bacteria (114) and higher animals (176) or to the formation of nuclear RNA. This nuclear RNA has mechanism of action of certain hormones (90), and could properties analogous to messenger RNA of bacteria, and be summarized as follows: an environmental stimulus, its inhibition by actinomycin D results in complete block perhaps a critical concentration of deoxynucleotides (48) big of DNA synthesis. In the 3rd stage, from 22 to 32 or a slight modification in the physical state of the DNA hr, the rate of RNA synthesis remains constant at its new molecule, may inactivate the repressor of a genetically in elevated level, and addition of actinomycin D does not active portion of DNA. The inactivation of the repressor result in inhibition of the subsequent DNA synthetic would cause the synthesis of a template RNA which, in period. In the same system, the concentration of such turn, would code the enzymes involved in the separation of enzymes as thymidine kinase and DNA polymerase in the DNA strands and the polymerization of the component creases only at the time DNA synthesis begins; (d) deoxynucleotides. In this context, it may be of interest to Giudice and Noveffi (80) found that the rise in DNA quote Swarm's conclusions (234) of a few years ago: polymerase that follows partial hepatectomy is inhibited â€œDifferentiationduring development is brought about by a by actinomycin D only when the antibiotic is given 3â€”8 process known as induction, the essence of which is that a hr after surgery, while puromycin (79), an inhibitor of short-lived stimulus of some sort produces a relatively

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1965 American Association for Cancer Research. 590 Cancer Research Vol. 25, June 1965 long-lasting effect on the pattern of synthesis. A number 11. Baserga, R., and Kisieleski, W. E. Cell Proliferation in Tu of lines of evidence suggest that stimulation of differen mor-bearing Mice. Arch. Pathol., 7@:142â€”48,1961. 12. . Comparative Studies of the Kinetics of Cellular tiated cells to divide is in fact an inductive process. . . .â€œ Proliferation of Normal and Tumorous Tissues with the Use of Tritiated Thymidine. I. Dilution of the Label and CONCLUSIONS Migration of Labeled Cells. J. Natl. Cancer Inst., 58: 331â€”39, 1962. In 1955, in a 12-page section dealing with cell division, 13. . Recent Observations on Cell Proliferation and Metab Cowdry (49) was able to condense our knowledge of cell olism by Radioautography with Tritiated Compounds. Atom interphase in a little less than 6 lines. Since then, the praxis, 8: 386-91, 1962. 14. . Effects of Histologic and Histochemical Procedures amount of information on metabolic activities of the inter on the Intensity of the Label in Radioautographs of Cells phase cell has reached such volume that a review of this Labeled with Triatiated Compounds. Lab. Invest., 1@: kind can only touch the most important points. There 648â€”56,1963. are still many uncertainties, but one is justified in saying 15. Baserga, R., Kisieleski, W. E., and Halvorsen, K. A Study that the life cycle of cells has been mapped out in its broad on the Establishment and Growth of Tumor Metastases with Tritiated Thymidine. Cancer Res., 50: 910â€”17,1960. outline, and that the details are being filled in rapidly. 16. Baserga, R., and Lisco, E. Duration of DNA Synthesis in Our knowledge of the cell cycle has had considerable Ehrlich Ascites Cells as Estimated by Double-Labeling with influence on 4 major areas of investigation: kinetics of Câ€•-and H'-Thymidine and Autoradiography. J. Nat!. Can cellular proliferation, control of DNA biosynthesis, life cer Inst., 31: 1559â€”71,1963. 17. Baserga, R., and Nemeroff, K. Two-Emulsion Radioautog span of cells, and cell differentiation. The last 2 areas raphy. J. Histochem. Cytochem., 10: 628â€”35,1962. have not been touched in the present review, but are 18. Baserga, R., Tyler, S. A., and Kisieleski, W. E. The Kinetics closely interrelated with cellular kinetics and cell division. of Growth of the Ehrlich Tumor. Arch. Pathol., 76: 9â€”13, For the student of cancer, it may be of interest to evaluate 1963. 19. Behki, R. M., and Schneider, W. C. Incorporation of Tn the impact exerted on our approach to the investigation of tiated Thymidine into Deoxyribonucleic Acid by Isolated cancer by knowledge of the cell cycle and by permanent Nuclei. Biochim. Biophys. Acta, 68: 34â€”44,1963. tagging of cells with DNA precursors. It is reasonable to 20. Belanger, L. F., and Leblond, C. P. A Method for Locat say that the kinetics of cellular proliferation, in normal as ing Radioactive Elements in Tissues by Covering Histo well as in tumorous tissues, have been made more truly logical Sections with a Photographic Emulsion. Endocrinol ogy,39:8â€”13,1946. quantitative and closer to the Pythagorean ideal that â€œthe 21. Bennett, L. L., Jr., Simpson, L., and Skipper, H. E. On the principles of mathematics are also the principles of things Metabolic Stability of Nucleic Acids in Mitotically Inactive as they areâ€•(196). 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Renato Baserga

Cancer Res 1965;25:581-595.

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